Fusion proteins for blood-brain barrier delivery

ABSTRACT

The invention provides compositions, methods, and kits for increasing transport of agents across the blood brain barrier while allowing their activity once across the barrier to remain substantially intact. The agents are transported across the blood brain barrier via one or more endogenous receptor-mediated transport systems. In some embodiments the agents are therapeutic, diagnostic, or research agents.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with the support of the United States governmentunder Grant number R44-NS-44654 by the National Institues of Health. TheUnited States Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Neurological disorders represent a major cause of mortality anddisability worldwide. Despite extensive progress, current treatmentoptions remain limited in some aspects. One major reason for thislimitation is that the brain is unique in allowing only select access tomolecules. While this is a useful protective mechanism, it also meansthat many potentially beneficial molecular entities do not have accessto the central nervous system (CNS), and thus are unable to exert atherapeutic effect in many neurological disorders or other conditions ofthe CNS. The present invention represents an advance in providingaccessibility of the CNS for molecular entities whose ability to crossthe blood brain barrier is limited.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compositions. In some embodiments,the invention provides a composition containing a neurotherapeutic agentcovalently linked to a structure that is capable of crossing the bloodbrain barrier (BBB), where the composition is capable of producing anaverage elevation of concentration in the brain of the neurotherapeuticagent of at least about 5 ng/gram brain following peripheraladministration. In some embodiments, the neurotherapeutic agent has amolecular weight greater than about 400. In some embodiments, theneurotherapeutic agent alone does not cross the BBB in a therapeuticallyeffective amount following peripheral administration. In someembodiments, the structure that is capable of crossing the BBB is astructure that crosses the BBB on an endogenous BBB receptor mediatedtransport system. In some embodiments, the endogenous BBB receptormediated transport system is the insulin receptor, transferrin receptor,leptin receptor, lipoprotein receptor, or the IGF receptor. In someembodiments, the endogenous BBB receptor mediated transport system isthe insulin BBB receptor mediated transport system. In some embodiments,the structure that is capable of crossing the BBB is an antibody, e.g.,a monoclonal antibody (MAb), such as a chimeric MAb. In someembodiments, the chimeric antibody contains sufficient human sequencesto avoid significant immunogenic reaction when administered to a human.In some embodiments, the neurotherapeutic agent is a neurotrophin. Insome embodiments, the neurotrophin is a brain derived neurotrophicfactor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblastgrowth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-1 receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, or stem cell factor (SCF). In some embodiments,the neurotrophin is brain derived neurotrophic factor (BDNF). In someembodiments, the BDNF is a variant of native BDNF, such as a two aminoacid carboxyl-truncated variant. In some embodiments, the BDNF is ahuman BDNF. In some embodiments, the BDNF contains a sequence that is atleast about 80% identical to the sequence of amino acids 466-582 of SEQID NO: 24. In some embodiments, the neurotherapeutic agent is aneurotrophin, e.g., BDNF and the structure that is capable of crossingthe blood brain barrier (BBB) is a MAb to an endogenous BBB receptormediated transport system, e.g., is an antibody to the insulin BBBreceptor mediated transport system. In some of these embodimentscontaining BDNF, the BDNF is a two amino acid carboxy-truncated BDNF. Insome of these embodiments containing a MAb, the MAb is a chimeric MAb,such as a chimeric antibody that contains sufficient human sequences toavoid significant immunogenic reaction when administered to a human.

In some embodiments, the invention provides a composition containing atwo-amino acid carboxy-truncated human BDNF covalently linked to achimeric MAb to the human insulin BBB receptor mediated transport systemcontaining sufficient human sequences to avoid significant immunogenicreaction when administered to a human, where the composition is capableof producing an average elevation of concentration in the brain of theneurotrophin of at least about 5 ng/gram brain following peripheraladministration, and where the BDNF contains a sequence that is at leastabout 80% identical to the sequence of amino acids 466-582 of SEQ ID NO:24. In some embodiments, the BDNF is covalently linked at its aminoterminus to the carboxy terminus of the heavy chain of the MAb. In someembodiments, the BDNF is covalently linked at its amino terminus to thecarboxy terminus of the light chain of the MAb. In some embodiments, theheavy chain of the MAb contains a sequence that is at least about 80%identical to amino acids 20-462 of SEQ ID NO: 24. In some embodiments,the composition further contains a linker between the heavy chain of theMAb and the BDNF, such as S—S-M. In some embodiments, the compositionfurther includes the light chain of the MAb. In some embodiments, thelight chain contains a sequence that is at least about 80% identical toamino acids 21-234 of SEQ ID NO: 36. In some embodiments, the MAb isglycosylated.

In some embodiments, the invention provides a pharmaceutical compositioncontaining any of the preceding compositions and a pharmaceuticallyacceptable excipient.

In some embodiments, the invention provides a composition containing acomposition that includes a neurotherapeutic agent covalently linked toa structure that is capable of crossing the blood brain barrier (BBB),where the composition is capable of producing an average elevation ofconcentration in the brain of the neurotherapeutic agent of at leastabout 5 ng/gram brain following peripheral administration; and furtherincludes a second composition containing a second neurotherapeutic agentcovalently linked to a second structure that is capable of crossing theblood brain barrier (BBB). In some embodiments, the first and secondneurotherapeutic agents are different and the first and secondstructures capable of crossing the BBB are the same structure. In someembodiments, the structure capable of crossing the BBB is an antibodycontaining a first heavy chain and a second heavy chain. In someembodiments, the first neurotherapeutic agent is covalently linked tothe first heavy chain of the antibody and the second neurotherapeuticagent is covalently linked to the second heavy chain of the antibody.

In some embodiments, the invention provides a composition containing anagent covalently linked to a chimeric MAb to the human BBB insulinreceptor, where the MAb contains a heavy chain and a light chain. Insome embodiments, the agent is a therapeutic agent. In some embodiments,the therapeutic agent is a neurotrophin, such as a BDNF. In someembodiments, the agent is a two amino acid carboxyl-terminal truncatedBDNF. In some embodiments, the heavy chain of the MAb is covalentlylinked to the BDNF to form a fusion protein, and the sequence of thefusion protein contains a first sequence that is at least about 80%identical to a sequence that includes amino acids 20-462 of SEQ ID NO:24 and further contains a second sequence that is at least about 80%identical to a sequence that includes amino acids 466-582 of SEQ ID NO:24; optionally, there may also be a peptide linker between the carboxylterminus of the first sequence and the amino terminus of the secondsequence, such as S—S-M. In some embodiments, the light chain of the MAbcontains a sequence that is at least about 80% identical to a sequencethat includes amino acids 21-234 of SEQ ID NO: 36. In some embodiments,the MAb is glycosylated.

In some embodiments, the invention provides a composition for treating aneurological disorder containing a BDNF covalently linked to animmunoglobulin that is capable of crossing the blood brain barrier,where the composition is capable of crossing the BBB in an amount thatis effective in treating the neurological disorder.

In some embodiments, the invention provides a fusion protein containing(i) a structure capable of crossing the BBB, covalently linked(optionally via a peptide linker) to (ii) a peptide that is active inthe central nervous system (CNS), where the structure capable ofcrossing the blood brain barrier and the peptide that is active in thecentral nervous system each retain an average of at least about 40% oftheir activities, compared to their activities as separate entities. Insome embodiments, the structure capable of crossing the blood brainbarrier crosses the BBB on an endogenous BBB receptor-mediatedtransporter. In some embodiments, the endogenous BBB receptor mediatedtransport system is the insulin receptor, transferrin receptor, leptinreceptor, lipoprotein receptor, or the IGF receptor. In someembodiments, the endogenous BBB receptor-mediated transporter is theinsulin transporter or the transferrin transporter. In some embodiments,the endogenous BBB receptor-mediated transporter is the insulintransporter, e.g., the human insulin transporter. In some embodiments,the structure capable of crossing the BBB is an antibody, e.g., a MAbsuch as a chimeric MAb. In some embodiments, the antibody is an antibodyto an endogenous BBB receptor-mediated transporter, such as the insulinreceptor, transferrin receptor, leptin receptor, lipoprotein receptor,or the IGF receptor, or such as the insulin transporter or thetransferrin transporter, or such as the insulin transporter, e.g., thehuman insulin transporter. In some embodiments, the peptide that isactive in the CNS is a neurotherapeutic agent. In some embodiments, theneurotherapeutic agent is a neurotrophin. In some embodiments, theneurotrophin is a brain derived neurotrophic factor (BDNF), nerve growthfactor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2 andother FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF. Insome embodiments, the BDNF is a truncated BDNF, e.g., carboxyl-truncatedBDNF, such as a BDNF that is lacking the two carboxyl terminal aminoacids.

In some embodiments, the invention provides a composition containing acationic therapeutic peptide covalently linked to an immunoglobulin,where the cationic therapeutic peptide in the composition has a serumhalf-life that is an average of at least about 5-fold greater than theserum half-life of the cationic therapeutic peptide alone. In someembodiments, the cationic therapeutic peptide contains aneurotherapeutic agent. In some embodiments, the neurotherapeutic agentis a neurotrophin. In some embodiments, the neurotrophin is brainderived neurotrophic factor (BDNF), nerve growth factor (NGF),neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs,neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor(HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF. Insome embodiments, the immunoglobulin is an antibody to an endogenous BBBreceptor-mediated transport system.

In another aspect, the invention provides methods. In one embodiment,the invention provides a method of transport of an agent active in theCNS from the peripheral circulation across the BBB in an effectiveamount, by peripherally administering to an individual the agentcovalently attached to a structure that crosses the BBB, underconditions where the agent covalently attached to a structure thatcrosses the BBB is transported across the BBB in an effective amount. Insome embodiments, the agent is a neurotherapeutic agent.

In some embodiments, the invention provides a method for treating a CNSdisorder in an individual, e.g., a human, by peripherally administeringto the individual an effective amount of a composition containing aneurotherapeutic agent covalently attached to a structure capable ofcrossing the BBB. In some embodiments, the structure capable of crossingthe BBB contains an antibody to an insulin receptor and the therapeuticagent comprises a BDNF. In some embodiments, the administering is oral,intravenous, intramuscular, subcutaneous, intraperitoneal, rectal,transbuccal, intranasal, transdermal, or inhalation administering. Insome embodiments, the administering is intravenous, intramuscular, orsubcutaneous. In some embodiments, the CNS disorder is an acute CNSdisorder, such as spinal cord injury, focal brain ischemia and globalbrain ischemia. In embodiments in which the disorder is an acutedisorder, in some embodiments the composition is administered only once.In embodiments in which the disorder is an acute disorder, in someembodiments the composition is administered at a frequency of no greaterthan about once per week. In some embodiments, the CNS disorder is achronic disorder. In some embodiments, the chronic disorder is selectedfrom the group consisting of chronic neurodegenerative disease, retinalischemia, or depression. In some embodiments where the disorder is achronic neurodegenerative disease, the chronic neurodegenerative diseaseis prion diseases, Alzheimer's disease, Parkinson's disease, amyotrophiclateral sclerosis, Huntington's disease, multiple sclerosis, transversemyelitis, motor neuron disease, Pick's disease, tuberous sclerosis,lysosomal storage disorders, Canavan's disease, Rett's syndrome,spinocerebellar ataxias, Friedreich's ataxia, optic atrophy, or retinaldegeneration. In some embodiments, e.g., where the individual is ahuman, the individual is administered a dose of the composition that isabout 1 to about 100 mg.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. Diagram showing genetic engineering of a eukaryotic expressionvector encoding a fusion gene comprised of the variable region of theheavy chain (VH) of the chimeric HIRMAb, a genomic fragment encoding theconstant region of human IgG1, which is comprised of 4 regions (CH1,hinge, CH2, and CH3), and the cDNA for the BDNF variant (vBDNF).Transcription of the gene is driven by the human IgG1 promoter (PRO).This vector produces the heavy chain (HC) of the fusion protein.

FIG. 2. Diagram showing genetic engineering of a bacterial expressionplasmid encoding vBDNF cDNA with modified 5′- and 3′-linkers.

FIG. 3. Ethidium bromide stained agarose gels showing size of variousconstructs that are intermediates in construction of a tandem vectorthat produces the fusion protein. (A) Lanes 1-2: plasmid from FIG. 2digested with NruI showing 0.4 kb vBDNF and 3.5 kb vector backbone. Lane3: MW size standards ranging from 1.4-0.1 kb. Lane 4: MW size standardsranging from 23-0.6 kb. (B) Lane 1: the 0.4 kb vBDNF cDNA is produced bythe polymerase chain reaction (PCR) using cDNA reverse transcribed frompolyA+RNA isolated from human U87 glioma cells; the PCR primer sequencesare given in Table 2. Lanes 2 and 3: same MW size standards as shown inpanel A. (C) lane 1: clone 416 following digestion with NheI and BamHI;lane 2: negative clone; lane 3: clone 400 following digestion with NheIand BamHI: lanes 4 and 5: same MW size standards as shown in panel A.(D) PCR fragments of DNA encoding fusion protein HC (lane 1) and LC(lane 2); lanes 3-4: same MW size standards as shown in panel A. (E)lanes 1-4: 4 different but identical copies of clone 422a followingdigestion with NheI, showing release of 0.4 kb fusion protein HCvariable region (VH) cDNA; lanes 5-6: same MW size standards as shown inpanel A. (F) lanes 1-4: 5 different but identical copies of clone 423afollowing digestion with EcoRV and BamHI, showing release of 0.7 kbentire LC cDNA; lanes 5-6: same MW size standards as shown in panel A.(G) Restriction endonuclease mapping of tandem vector (FIG. 12) withPvuI (lane 1), and EcoRI-HindIII (lane 2). PvuI (single cut) producedthe expected linear DNA band of ˜11 kb. Digestion with EcoRI and HindIIIreleases both the fusion protein light chain (i.e. 1.8 kb) and DHFR(i.e. 1.5 kb) expression cassettes. The ˜8 kb band represents thebackbone vector with the fusion protein heavy chain expression cassette;lanes 3-4: same MW size standards as shown in panel A, albeit in reverseorder.

FIG. 4. Nucleotide (SEQ ID NO: 21) and amino acid (SEQ ID NO: 22)sequence of fusion site between carboxyl terminus of the fusion proteinHC and the amino terminus of the vBDNF. The 3-amino acid linker betweenthe HIRMAb HC and the vBDNF is shown, as well as the new stop codon atthe carboxyl terminus of vBDNF.

FIG. 5. Nucleotide sequence (SEQ ID NO: 23) of fusion protein HC genecloned into plasmid 416. Italics: human IgG1 constant region introns;bold font: human IgG1 exon sequence; underline font: vBDNF.

FIG. 6. Amino acid sequence (SEQ ID NO: 24) of the fusion protein HC.The 19 amino acid signal peptide is underlined, as is the 3-amino acidlinker between the CH3 region and the vBDNF. The N-linked glycosylationconsensus sequence within CH2 is underlined.

FIG. 7. The amino acid sequence (SEQ ID NO: 25) of the different domainsof the fusion protein HC are shown.

FIG. 8. Diagram showing production of the intronless eukaryoticexpression vector, clone 422a, which encodes the fusion protein HC. Thefusion protein HC cDNA was produced by PCR from cDNA generated byreverse transcriptase of RNA isolated from myeloma cells transfectedwith clone 416.

FIG. 9. (A) Nucleotide sequence (SEQ ID NO: 26) of the fusion protein HCcDNA inserted in clone 422a. (B) (SEQ ID NOS 27 & 28) Amino acidsequence of the fusion protein HC that is deduced from the nucleotidesequence shown in panel A. The sequence of the signal peptide isunderlined.

FIG. 10. Diagram showing production of the intronless eukaryoticexpression vector, clone 423a, which encodes the fusion protein LC. Thefusion protein LC cDNA was produced by PCR from cDNA generated byreverse transcriptase of RNA isolated from myeloma cells transfectedwith an expression vector producing the LC gene that was derived fromchromosomal fragment encoding intron/exon sequence of the human kappa LCgene with the VL of the chimeric HIRMAb LC.

FIG. 11. (A) Nucleotide sequence (SEQ ID NO: 29) of the fusion proteinLC cDNA inserted in clone 423a. (B) (SEQ ID NOS 30 & 31) Amino acidsequence of the fusion protein LC that is deduced from the nucleotidesequence shown in panel A. The sequence of the signal peptide isunderlined.

FIG. 12. Diagram showing the construction of a tandem vector encodingthe HC and LC genes of the fusion protein. The TV was engineered fromthe cDNA expression vectors, clones 422a and 423a, for the HC and LC,respectively, as well as from a bacterial expression plasmid encodingthe expression cassette for mouse DHFR.

FIGS. 13A and 13B. Nucleotide sequence (SEQ ID NO: 32) of the fusionprotein HC gene and LC gene, and the DHFR genes incorporated in thetandem vector.

FIG. 14. Deduced amino acid sequence of the fusion protein HC based ontandem vector nucleotide sequence analysis (SEQ ID NOS 33 & 34). Thesignal peptide sequence is underlined.

FIG. 15. Deduced amino acid sequence of the fusion protein LC based ontandem vector nucleotide sequence analysis (SEQ ID NO 35 & 36). Thesignal peptide sequence is underlined.

FIG. 16. Deduced amino acid sequence of the DHFR based on tandem vectornucleotide sequence analysis (SEQ ID NO 37 & 38).

FIG. 17. Viable and total cell density of CHO cells in bioreactormaintained continuously for 50 days; the CHO cells had been permanentlytransfected with the tandem vector encoding the fusion protein.

FIG. 18. Structure of fusion protein, a bi-functional molecule that both(a) binds to the human BBB human insulin receptor (HIR) to enabletransport across the BBB from blood, and (b) binds to the trkB onneurons to induce neuroprotection.

FIG. 19. Correlation of 2 different ‘sandwich’ immunoassays, where thesecondary antibody is either directed against the Fc region of humanIgG1 (x-axis) or against human BDNF (y-axis). The primary antibody ineither assay is directed against the human kappa light chain. Themeasured level of fusion protein in CHO cell conditioned medium is thesame whether the anti-Fc or the anti-BDNF antibody is used.

FIG. 20. Reducing (left) and non-reducing (right) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) of chimeric HIRMAb andfusion protein. Under reducing conditions, the size of the light chain,30 kDa, is identical for chimeric HIRMAb and the fusion protein; thesize of the heavy chain of fusion protein is about 15 kDa larger thanthe chimeric HIRMAb heavy chain, owing to the presence of the BDNF.Under non-reducing conditions, the chimeric HIRMAb and the fusionprotein migrate as single hetero-tetrameric species with molecularweights of 180 and 200 kDa, respectively.

FIG. 21. (Left panel) Western blot with anti-human IgG primary antibody.The size of the heavy chain of the fusion protein and the chimericHIRMAb is 64 kDa and 50 kDa, respectively, and the size of the lightchain for either the fusion protein or the chimeric HIRMAb is 25 kDa.(Right panel) Western blot with anti-human BDNF antibody, which reactswith either fusion protein or BDNF, but not with chimeric HIRMAb. MWstandards (STDS) are shown on the right side.

FIG. 22. Isoelectric focusing (IEF) of isoelectric point (pI) standards(lane 1), chimeric HIRMAb (lanes 2 and 4), BDNF (lane 3), and fusionprotein (lane 5). Whereas BDNF is highly cationic with a pI>10, the pIof the fusion protein approximates the pI of the chimeric HIRMAb, whichis about 8.5, and close to the theoretical pI of the fusion protein.

FIG. 23. (A) Outline for human insulin receptor (HIR) competitive ligandbinding assay (CLBA). The HIR extracellular domain (ECD) is bound by the[¹²⁵I]-labeled murine HIRMAb, and this binding is competitivelydisplaced by either the chimeric HIRMAb or the fusion protein, as shownin Panel B. (B) Displacement of binding of [¹²⁵I]-labeled murine HIRMAbto the HIR ECD by either chimeric HIRMAb or fusion protein. The affinityof the chimeric HIRMAb to the HIR ECD is high, and the affinity of thefusion protein for the HIR ECD is not significantly different from thatof the chimeric HIRMAb. These results show that the fusion of the vBDNFto the carboxyl terminus of the chimeric HIRMAb heavy chain does notimpair binding of the fusion protein to the HIR.

FIG. 24. (A) Design of trkB competitive ligand binding assay (CLBA). Theadvantage of the PEG linker is that this modification eliminates thehigh non-specific binding (NSB) of the cationic BDNF to the ELISA wells,which gives an assay with a high signal/noise ratio. The binding of theBDNF-PEG²⁰⁰⁰-biotin to the trkB ECD was detected with a peroxidasesystem using avidin and biotinylated peroxidase. (B) The binding of theBDNF-PEG²⁰⁰⁰-biotin to the trkB ECD is competitively displaced byrecombinant BDNF. This binding data was analyzed by non-linearregression analysis to yield the K₁ of BDNF binding, 3.5±1.3 pmol/welland the NSB parameter. (C) The binding of the BDNF-PEG²⁰⁰⁰-biotin to thetrkB ECD is competitively displaced by the fusion protein. This bindingdata was analyzed by non-linear regression analysis to yield the K₁ offusion protein binding, 2.2±1.2 pmol/well, which is not significantlydifferent than the K₁ for native BDNF. These data show that the affinityof the fusion protein for the trkB receptor is equal to that of nativeBDNF.

FIG. 25. (A) Design of hypoxia-reoxygenation neuroprotection assay inhuman neural SH-SY5Y cells. Exposure of the cells to retinoic acid for 7days causes an up-regulation in the gene expression of trkB, the BDNFreceptor. (B) Neuroprotection assay based on the measurement ofmitochondrial respiration with3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT).The maximal neuroprotection is established with 4 nM BDNF, and 4 nMfusion protein yields a comparable level of neuroprotection in humanneural cells. The MTT level does not return to that of non-hypoxiccells, because only about 50% of the cells induce trkB in response toretinoic acid.

FIG. 26. (A) Light micrograph of capillaries isolated from human brain,used as an in vitro model system of the human BBB. (B) Radio-receptorassay of binding of [³H]-fusion protein to the HIR on the human BBB; thebinding is self-inhibited by unlabeled fusion protein. Fitting thesaturation data to a Scatchard plot with a non-linear regressionanalysis yields the binding parameters: K_(D)=0.55±0.07 nM,B_(max)=1.35±0.10 pmol/mg_(p).

FIG. 27. Pharmacokinetics and brain uptake of fusion protein in theadult Rhesus monkey. (A) The serum concentration of [³H]-fusion protein,or [¹²⁵I]-murine HIRMAb, is plotted vs. time after a single intravenousinjection of either protein in anesthetized adult Rhesus monkeys. (B)The serum radioactivity that is precipitable by trichloroacetic acid(TCA) is plotted vs time after a single intravenous injection of either[³H]-fusion protein in the anesthetized adult Rhesus monkey, or[³H]-BDNF in the anesthetized adult rat. (C) Capillary depletionanalysis of brain distribution at 180 minutes after a single intravenousinjection of either [³H]-fusion protein, or [³H]-mouse IgG2a, in theanesthetized adult Rhesus monkey. (D) Primate brain concentrations offusion protein at 180 minutes after an intravenous injection of 373 μgfusion protein, as compared to the endogenous primate brainconcentration of BDNF.

DETAILED DESCRIPTION OF THE INVENTION

-   I. Introduction-   II. Definitions-   III. The blood brain barrier

A. Transport systems

B. Structures that bind to a blood brain barrier receptor-mediatedtransport system

-   IV. Agents for transport across the blood brain barrier

A. Neurotrophins

B. Brain-derived neurotrophic factor

-   V. Compositions-   VI. Nucleic acids, vecors, cells, and manufacture

A. Nucleic acids

B. Vectors

C. Cells

D. Manufacture

-   VII. Methods-   VIII. Kits

Abbreviations

-   ALS amyotrophic lateral sclerosis-   BBB blood-brain barrier-   BDNF brain derived neurotrophic factor-   BRB blood-retinal barrier-   CDR complementarity determining region-   CED convection enhanced diffusion-   CHI first part of IgG constant region-   CH2 second part of IgG constant region-   CH3 third part of IgG constant region-   CHO Chinese hamster ovarian cell-   CHOP CHO cell host protein-   CLBA competitive ligand binding assay-   CMV cytomegalovirus-   CNS central nervous system-   DHFR dihydrofolate reductase-   ECD extracellular domain-   FR framework region-   FWD forward-   HIR human insulin receptor-   HIRMAb monoclonal antibody to human insulin receptor-   HIV human immune deficiency virus-   IC intra-cerebral-   ICC immunocytochemistry-   ICV intra-cerebroventricular-   ID injected dose-   IEF isoelectric focusing-   IGF insulin-like growth factor-   IgG immunoglobulin G-   LDL low density lipoprotein-   MAb monoclonal antibody-   MRT mean residence time-   MTH molecular Trojan horse-   MTX methotrexate-   MW molecular weight-   NSB non-specific binding-   NT-3 neurotrophin-3-   NT4/5 neurotrophin-4/5-   ODN oligodeoxynucleotide-   PEG polyethyleneglycol-   PRO promoter-   REV reverse-   RMT receptor mediated transport-   SDM site-directed mutagenesis-   SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis-   SFM serum free medium-   SNP single nucleotide polymorphism-   TCA trichloroacetic acid-   TFF tangential flow filtration-   TFI transient forebrain ischemia-   TfR transferrin receptor-   TrkB BDNF receptor-   TV tandem vector-   vBDNF variant of BDNF-   V_(D) volume of distribution-   VH variable region of heavy chain-   VL variable region of light chain

I. INTRODUCTION

The blood brain barrier is a limiting factor in the delivery of manyperipherally-administered agents to the central nervous system. Thepresent invention addresses three factors that are important indelivering an agent across the BBB to the CNS: 1) A pharmacokineticprofile for the agent that allows sufficient time in the peripheralcirculation for the agent to have enough contact with the BBB totraverse it; 2) Modification of the agent to allow it to cross the BBB;and 3) Retention of activity of the agent once across the BBB. Variousaspects of the invention address these factors, by providing fusionstructures (e.g., fusion proteins) of an agent (e.g., a therapeuticagent) covalently linked to a structure that causes the agent to haveincreased serum half life, to be transported across the BBB, and/or toretain some or all of its activity in the brain while still attached tothe structure.

Accordingly, in one aspect, the invention provides compositions andmethods that utilize an agent covalently linked to a structure capableof crossing the blood brain barrier (BBB). The compositions and methodsare useful in transporting agents, e.g. therapeutic agents such asneurotherapeutic agents, from the peripheral blood and across the BBBinto the CNS. Neurotherapeutic agents useful in the invention includeneurotrophins, e.g. brain-derived neurotrophic factor (BDNF). In someembodiments, the structure that is capable of crossing the BBB iscapable of binding to an endogenous BBB receptor mediated transportsystem and crossing the BBB. In some embodiments, the structure that iscapable of crossing the BBB is an antibody, e.g., a monoclonal antibody(MAb) such as a chimeric MAb.

In some embodiments, the invention provides a fusion protein thatincludes a structure capable of crossing the BBB covalently linked to apeptide that is active in the central nervous system (CNS), where thestructure capable of crossing the blood brain barrier and the peptidethat is active in the central nervous system each retain a proportion(e.g., 10-100%) of their activities (or their binding affinities fortheir respective receptors), compared to their activities (or theirbinding affinities for their respective receptors) as separate entities.

In another aspect, the invention provides a composition containing acationic therapeutic peptide covalently linked to an immunoglobulin,where the cationic therapeutic peptide in the composition has a serumhalf-life that is an average of at least about 5-fold greater than theserum half-life of the cationic therapeutic peptide alone.

The invention also provides nucleic acids coding for peptides andproteins. In some embodiments, the invention provides a single nucleicacid sequence that contains a gene coding for a light chain of animmunoglobulin and a gene coding for a fusion protein made up of a heavychain of the immunoglobulin covalently linked to a peptide. In someembodiments the peptide of the fusion protein is a therapeutic peptide,e.g., a neurotherapeutic peptide such as a neurotrophin. The inventionalso provides vectors containing the nucleic acids of the invention, andcells containing the vectors. Further provided are methods ofmanufacturing an immunoglobulin fusion protein, where the fusion proteincontains an immunoglobulin heavy chain fused to a therapeutic agent,where the methods include permanently integrating into a eukaryotic cella single tandem expression vector in which both the immunoglobulin lightchain gene and the gene for the immunoglobulin heavy chain fused to thetherapeutic agent are incorporated into a single piece of DNA.

The invention further provides therapeutic compositions, such aspharmaceutical compositions that contain an agent covalently linked to astructure capable of crossing the blood brain barrier (BBB) and apharmaceutically acceptable excipient. In some embodiments, theinvention provides a composition for treating a neurological disorderthat includes a BDNF covalently linked to an immunoglobulin that iscapable of crossing the blood brain barrier, wherein the composition iscapable of crossing the BBB in an amount that is effective in treatingthe neurological disorder.

The invention also provides methods for treating a neurological disorderin an individual that include peripherally administering to theindividual an effective amount of one or more of the compositions of theinvention, optionally in combination with other therapy for thedisorder.

H. DEFINITIONS

As used herein, an “agent” includes any substance that is useful inproducing an effect, including a physiological or biochemical effect inan organism. A “therapeutic agent” is a substance that produces or isintended to produce a therapeutic effect, i.e., an effect that leads toamelioration, prevention, and/or complete or partial cure of a disorder.A “therapeutic effect,” as that term is used herein, also includes theproduction of a condition that is better than the average or normalcondition in an individual that is not suffering from a disorder, i.e.,a supranormal effect such as improved cognition, memory, mood, or othercharacteristic attributable at least in part to the functioning of theCNS, compared to the normal or average state. A “neurotherapeutic agent”is an agent that produces a therapeutic effect in the CNS. A“therapeutic peptide” includes therapeutic agents that consists of apeptide. A “cationic therapeutic peptide” encompasses therapeuticpeptides whose isoelectric point is above about 7.4, in someembodiments, above about 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, orabove about 12.5. A subcategory of cationic therapeutic peptides iscationic neurotherapeutic peptides.

As used herein, a “peptide that is active in the central nervous system(CNS)” includes peptides that have an effect when administered to theCNS. The effect may be a therapeutic effect or a non-therapeutic effect,e.g., a diagnostic effect or an effect useful in research. If the effectis a therapeutic effect, then the peptide is also a therapeutic peptide.A therapeutic peptide.that is also a peptide that is active in the CNSis encompassed by the term “neurotherapeutic peptide,” as used herein.

“Treatment” or “treating” as used herein includes achieving atherapeutic benefit and/or a prophylactic benefit. By therapeuticbenefit is meant eradication or amelioration of the underlying disorderor condition being treated. For example, in an individual with aneurological disorder, therapeutic benefit includes partial or completehalting of the progression of the disorder, or partial or completereversal of the disorder. Also, a therapeutic benefit is achieved withthe eradication or amelioration of one or more of the physiological orpsychological symptoms associated with the underlying condition suchthat an improvement is observed in the patient, notwithstanding the factthat the patient may still be affected by the condition. A prophylacticbenefit of treatment includes prevention of a condition, retarding theprogress of a condition (e.g., slowing the progression of a neurologicaldisorder), or decreasing the likelihood of occurrence of a condition. Asused herein, “treating” or “treatment” includes prophylaxis.

As used herein, the term “effective amount” can be an amount sufficientto effect beneficial or desired results, such as beneficial or desiredclinical results, or enhanced cognition, memory, mood, or other desiredCNS results. An effective amount is also an amount that produces aprophylactic effect, e.g., an amount that delays, reduces, or eliminatesthe appearance of a pathological or undesired condition. Such conditionsof the CNS include dementia, neurodegenerative diseases as describedherein, suboptimal memory or cognition, mood disorders, general CNSaging, or other undesirable conditions. An effective amount can beadministered in one or more administrations. In terms of treatment, an“effective amount” of a composition of the invention is an amount thatis sufficient to palliate, ameliorate, stabilize, reverse or slow theprogression of a disorder, e.g., a neurological disorder. An “effectiveamount” may be of any of the compositions of the invention used alone orin conjunction with one or more agents used to treat a disease ordisorder. An “effective amount” of a therapeutic agent within themeaning of the present invention will be determined by a patient'sattending physician or veterinarian. Such amounts are readilyascertained by one of ordinary skill in the art and will a therapeuticeffect when administered in accordance with the present invention.Factors which influence what a therapeutically effective amount will beinclude, the specific activity of the therapeutic agent being used, thetype of disorder (e.g., acute vs. chronic neurological disorder), timeelapsed since the initiation of the disorder, and the age, physicalcondition, existence of other disease states, and nutritional status ofthe individual being treated. Additionally, other medication the patientmay be receiving will affect the determination of the therapeuticallyeffective amount of the therapeutic agent to administer.

A “subject” or an “individual,” as used herein, is an animal, forexample, a mammal. In some embodiments a “subject” or an “individual” isa human. In some embodiments, the subject suffers from a neurologicaldisorder.

In some embodiments, an agent is “administered peripherally” or“peripherally administered.” As used herein, these terms refer to anyform of administration of an agent, e.g., a therapeutic agent, to anindividual that is not direct administration to the CNS, i.e., thatbrings the agent in contact with the non-brain side of the blood-brainbarrier. “Peripheral administration,” as used herein, includesintravenous, subcutaneous, intramuscular, intraperitoneal, transdermal,inhalation, transbuccal, intranasal, rectal, and oral administration.

A “pharmaceutically acceptable carrier” or “pharmaceutically acceptableexcipient” herein refers to any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Such carriers are well known to those of ordinary skill inthe art. A thorough discussion of pharmaceutically acceptablecarriers/excipients can be found in Remington's Pharmaceutical Sciences,Gennaro, A R., ed., 20th edition, 2000: Williams and Wilkins PA, USA.Exemplary pharmaceutically acceptable carriers can include salts, forexample, mineral acid salts such as hydrochlorides, hydrobromides,phosphates, sulfates, and the like; and the salts of organic acids suchas acetates, propionates, malonates, benzoates, and the like. Forexample, compositions of the invention may be provided in liquid form,and formulated in saline based aqueous solution of varying pH (5-8),with or without detergents such polysorbate-80 at 0.01-1%, orcarbohydrate additives, such mannitol, sorbitol, or trehalose. Commonlyused buffers include histidine, acetate, phosphate, or citrate.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally occurring amino acid, e.g., an amino acid analog. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell. Probes8:91-98 (1994)).

The terms “isolated” and “purified” refer to a material that issubstantially or essentially removed from or concentrated in its naturalenvironment. For example, an isolated nucleic acid may be one that isseparated from the nucleic acids that normally flank it or other nucleicacids or components (proteins, lipids, etc. . . . ) in a sample. Inanother example, a polypeptide is purified if it is substantiallyremoved from or concentrated in its natural environment. Methods forpurification and isolation of nucleic acids and peptides are well knownin the art.

III. THE BLOOD BRAIN BARRIER

In one aspect, the invention provides compositions and methods thatutilize an agent covalently linked to a structure capable of crossingthe blood brain barrier (BBB). The compositions and methods are usefulin transporting agents, e.g. therapeutic agents such as neurotherapeuticagents, from the peripheral blood and across the blood brain barrierinto the CNS. As used herein, the “blood-brain barrier” refers to thebarrier between the peripheral circulation and the brain and spinal cordwhich is formed by tight junctions within the brain capillaryendothelial plasma membranes, creates an extremely tight barrier thatrestricts the transport of molecules into the brain, even molecules assmall as urea, molecular weight of 60 Da. The blood-brain barrier withinthe brain, the blood-spinal cord barrier within the spinal cord, and theblood-retinal barrier within the retina, are contiguous capillarybarriers within the central nervous system (CNS), and are collectivelyreferred to as the blood-brain barrier or BBB.

The BBB is a limiting step in the development of new neurotherapeutics,diagnostics, and research tools for the brain and CNS. Essentially 100%of large molecule therapeutics such as recombinant proteins, antisensedrugs, gene medicines, monoclonal antibodies, or RNA interference(RNAi)-based drugs, do not cross the BBB in pharmacologicallysignificant amounts. While it is generally assumed that small moleculedrugs can cross the BBB, in fact, <2% of all small molecule drugs areactive in the brain owing to the lack transport across the BBB. Amolecule must be lipid soluble and have a molecular weight less than 400Daltons (Da) in order to cross the BBB in pharmacologically significantamounts, and the vast majority of small molecules do not have these dualmolecular characteristics. Therefore, most potentially therapeutic,diagnostic, or research molecules do not cross the BBB inpharmacologically active amounts. So as to bypass the BBB, invasivetranscranial drug delivery strategies are used, such asintracerebro-ventricular (ICV) infusion, intracerebral (IC)administration, and convection enhanced diffusion (CED). Transcranialdrug delivery to the brain is expensive, invasive, and largelyineffective. The ICV route delivers BDNF only to the ependymal surfaceof the brain, not into brain parenchyma, which is typical for drugsgiven by the ICV route. The IC administration of a neurotrophin, such asnerve growth factor (NGF), only delivers drug to the local injectionsite, owing to the low efficiency of drug diffusion within the brain.The CED of neurotrophin results in preferential fluid flow through thewhite matter tracts of brain, which causes demyelination, andastrogliosis.

The present invention offers an alternative to these highly invasive andgenerally unsatisfactory methods for bypassing the BBB, allowing agents,e.g., neuroprotective factors to cross the BBB from the peripheralblood. It is based on the use of endogenous transport systems present inthe BBB to provide a mechanism to transport a desired substance from theperipheral blood to the CNS.

A. Transport Systems

In some embodiments, the invention provides compositions that include astructure that binds to a BBB receptor mediated transport system,coupled to an agent.for which transport across the BBB is desired, e.g.,a neurotherapeutic agent. The cornpositions and methods of the inventionmay utilize any suitable structure that is capable of transport by theselected endogenous BBB receptor-mediated transport system, and that isalso capable of attachment to the desired agent. In some embodiments,the structure is an antibody. In some embodiment the antibody is amonoclonal antibody (MAb), e.g., a chimeric MAb.

Endogenous BBB receptor-mediated transport systems The BBB has beenshown to have specific receptors that allow the transport from the bloodto the brain of several macromolecules; these transporters are suitableas transporters for compositions of the invention. Endogenous BBBreceptor-mediated transport systems useful in the invention includethose that transport insulin, transferrin, insulin-like growth factors 1and 2 (IGF1 and IGF2), leptin, and lipoproteins. In some embodiments,the invention utilizes a structure that is capable of crossing the BBBvia the endogenous insulin BBB receptor-mediated transport system, e.g.,the human endogenous insulin BBB receptor-mediated transport system.

B. Structures That Bind to a BBB Receptor Mediated Transport System

One noninvasive approach for the delivery of drugs to the CNS is toattach the agent of interest to a structure, e.g., molecule that bindswith receptors on the BBB. The structure then serves as a vector fortransport of the agent across the BBB. Such structures are referred toherein as “molecular Trojan horses (MTH).” Typically, though notnecessarily, a MTH is an exogenous peptide or peptidomimetic moiety(e.g., a MAb) capable of binding to an endogenous BBB receptor mediatedtransport system that traverses the BBB on the endogenous BBBreceptor-mediated transport system. In certain embodiments, the MTH canbe an antibody to a receptor of the transport system, e.g., the insulinreceptor. In some embodiments, the antibody is a monoclonal antibody(MAb). In some embodiments, the MAb is a chimeric MAb. Thus, despite thefact that Abs normally are excluded from the brain, they can be aneffective vehicle for the delivery of molecules into the brainparenchyma if they have specificity for receptors on the BBB.

Accordingly, antibodies are particularly useful in embodiments of theinvention, especially MAbs. Certain receptor-specific MAbs may mimic theendogenous ligand and function as a MTH and traverse a plasma membranebarrier via transport on the specific receptor system. In certainembodiments, the MTH is a MAb to the human insulin receptor (HIR) on thehuman BBB. The HIR MAb binds an exofacial epitope on the human BBB HIRand this binding enables the MAb to traverse the BBB via a transportreaction that is mediated by the human BBB insulin receptor.

An “antibody,” as that term is used herein, includes reference to anymolecule, whether naturally-occurring, artificially induced, orrecombinant, which has specific immunoreactive activity. Generally,though not necessarily, an antibody is a protein that includes twomolecules, each molecule having two different polypeptides, the shorterof which functions as the light chains of the antibody and the longer ofwhich polypeptides function as the heavy chains of the antibody.Normally, as used herein, an antibody will include at least one variableregion from a heavy or light chain. Additionally, the antibody maycomprise combinations of variable regions. The combination may includemore than one variable region of a light chain or of a heavy chain. Theantibody may also include variable regions from one or more light chainsin combination with variable regions of one or more heavy chains. Anantibody can be an immunoglobulin molecule obtained by in vitro or invivo generation of the humoral response, and includes both pblyclonaland monoclonal antibodies. Furthermore, the present invention includesantigen binding fragments of the antibodies described herein, such asFab, Fab′, F(ab)₂, and Fv fragments, fragments comprised of one or moreCDRs, single-chain antibodies (e.g., single chain Fv fragments (scFv)),disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies(e.g., bispecific antibodies), pFv fragments, heavy chain monomers ordimers, light chain monomers or dimers, and dimers consisting of oneheavy chain and one light chain. Such antibody fragments may be producedby chemical methods, e.g., by cleaving an intact antibody with aprotease, such as pepsin or papain, or via recombinant DNA techniques,e.g., by using host cells transformed with truncated heavy and/or lightchain genes. Synthetic methods of generating such fragments are alsocontemplated. Heavy and light chain monomers may similarly be producedby treating an intact antibody with a reducing agent, such asdithiothreitol or beta.-mercaptoethanol, or by using host cellstransformed with DNA encoding either the desired heavy chain or lightchain or both. An antibody immunologically reactive with a particularantigen can be generated in vivo or by recombinant methods such asselection of libraries of recombinant antibodies in phage or similarvectors.

A “chimeric” antibody includes an antibody derived from a combination ofdifferent mammals. The mammal may be, for example, a rabbit, a mouse, arat, a goat, or a human. The combination of different mammals includescombinations of fragments from human and mouse sources.

In some embodiments, an antibody of the present invention is amonoclonal antibody (MAb), typically a human monoclonal antibody. Suchantibodies are obtained from transgenic mice that have been “engineered”to produce specific human antibodies in response to antigenic challenge.In this technique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas.

For use in. humans, a chimeric MAb is preferred that contains enoughhuman sequence that it is not significantly immunogenic whenadministered to humans, e.g., about 80% human and about 20% mouse, orabout 85% human and about 15% mouse, or about 90% human and about 10%mouse, or about 95% human and 5% mouse, or greater than about 95% humanand less than about 5% mouse. Chimeric antibodies to the human BBBinsulin receptor with sufficient human sequences for use in theinvention are described in, e.g., Coloma et al. (2000) Pharm. Res. 17:266-274, which is incorporated by reference herein in its entirety. Amore highly humanized form of the HIR MAb can also be engineered, andthe humanized HIRMAb has activity comparable to the murine HIRMAb andcan be used in embodiments of the invention. See, e.g., U.S. PatentApplication Publication No. 20040101904, filed Nov. 27, 2002,incorporated by reference herein in its entirety.

Antibodies used in the invention may be glycosylated ornon-glycosylated. If the antibody is glycosylated, any pattern ofglycosylation that does not significantly affect the function of theantibody may be used. Glycosylation can occur in the pattern typical ofthe cell in which the antibody is made, and may vary from cell type tocell type. For example, the glycosylation pattern of a monoclonalantibody produced by a mouse myeloma cell can be different than theglycosylation pattern of a monoclonal antibody produced by a transfectedChinese hamster ovary (CHO) cell. In some embodiments, the antibody isglycosylated in the pattern produced by a transfected Chinese hamsterovary (CHO) cell.

Accordingly, in some embodiments, a genetically engineered HIR MAb, withthe desired level of human sequences, is fused to an agent for whichtransport across the BBB is desired, e.g. a neurotherapeutic agent suchas a neurotrophin such as human BDNF, to produce a recombinant fusionprotein that is a bi-functional molecule. The recombinant therapeuticneuroprotective factor/HIRMAb is able to both (i) cross the human BBB,via transport on the BBB HIR, and (ii) activate the factor's target,e.g., neuronal BDNF receptor, trkB, to cause neurotherapeutic effectsonce inside the brain, following peripheral administration.

IV. AGENTS FOR TRANSPORT ACROSS THE BBB

The agent for which transport across the BBB is desired may be anysuitable substance for introduction into the CNS. Generally, the agentis a substance for which transport across the BBB is desired, which doesnot, in its native form, cross the BBB in significant amounts. The agentmay be, e.g., a therapeutic agent, a diagnostic agent, or a researchagent. Diagnostic agents include peptide radiopharmaceuticals, such asthe epidermal growth factor (EGF) for imaging brain cancer (Kurihara andPardridge (1999) Canc. Res. 54: 6159-6163), and amyloid peptides forimaging brain amyloid such as in Alzheimers disease (Lee et al (2002) J.Cereb. Blood Flow Metabol. 22: 223-231). In some embodiments, the agentis a therapeutic agent, such as a neurotherapeutic agent. Apart fromneurotrophins, potentially useful therapeutic protein agents includerecombinant enzymes for lysosomal storage disorders (see, e.g., U.S.Patent Application Publication No. 20050142141, filed Feb. 17, 2005,incorporated by reference herein in its entirety), monoclonal antibodiesthat either mimic an endogenous peptide or block the action of anendogenous peptide, polypeptides for brain disorders, such as secretinfor autism (Ratliff-Schaub et al (2005) Autism 9: 256-265), opioidpeptides for drug or alchol addiction (Cowen et al, (2004) J. Neurochem.89: 273-285), or neuropeptides for apetite control (Jethwa et al (2005)Am. J. Physiol. 289: E301-305). In some embodiments, the agent is aneurotrophic factor, also referred to herein as a “neurotrophin.” Thus,in some embodiments, the invention provides compositions and methodsthat utilize a neurotrophin. In some embodiments, a single neurotrophinmay be used. In others, combinations of neurotrophins are used. In someembodiments, the invention utilizes a brain-derived neurotrophic factor(BDNF).

A. Neurotrophins

Many neurotrophic factors are neuroprotective in brain, but do not crossthe blood-brain barrier. These factors are suitable for use in thecompositions and methods of the invention and include brain-derivedneurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5,fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-l receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, and stem cell factor (SCF). Particularly usefulin some embodiments of the invention utilizing neurotrophins that areused as precursors for fusion proteins that cross the BBB are those thatnaturally form dimeric structures, similar to BDNF. Certainneurotrophins such as BDNF or NT-3 may form hetero-dimeric structures,and in some embodiments the invention provides a fusion proteinconstructed of one neurotrophin monomer fused to one chain (e.g., alight or heavy chain) of an antibody, e.g., of the HIRMAb, and anotherneurotrophin monomer fused to the second chain (e.g., a light or heavychain) of the antibody. Typically, the molecular weight range ofrecombinant proteins that may be fused to the molecular Trojan horseranges from 1000 Daltons to 500,000 Daltons.

B. Brain-Derived Neurotrophic Factor

One particularly useful neurotrophin in embodiments of the invention isbrain-derived neurotrophic factor (BDNF). BDNF is a powerfulneurotherapeutic that can be used as a neuroprotective agent in manyacute and chronic brain diseases. However, the lack of transport of BDNFacross the BBB has prevented the development of this molecule as aneurotherapeutic for the brain and spinal cord.

BDNF is a neurotherapeutic that is useful for the treatment of acute andchronic brain disease. In experimental stroke, the intracerebraladministration of BDNF is neuroprotective. In global brain ischemia,such as might follow a cardiac arrest, the direct intracerebraladministration of BDNF is neuroprotective. In experimental models ofchronic neurodegenerative disease such as prion diseases, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD), oramyotrophic lateral sclerosis (ALS), the direct intracerebral injectionof BDNF is neuroprotective.

In studies demonstrating the pharmacologic efficacy of BDNF inexperimental brain disease, it is necessary to administer theneurotrophin directly into the brain following a transcranial drugdelivery procedure. The transcranial drug delivery is required becauseBDNF does not cross the brain capillary wall, which forms theblood-brain barrier (BBB) in vivo. Owing to the lack of transport ofBDNF across the BBB, it is not possible for the neurotrophin to enterthe CNS, including the brain or spinal cord, following a peripheraladministration unless the BBB is experimentally disrupted. Clinicaltrials showed that subcutaneous administration of BDNF was not effectivein the treatment of chronic neurodegenerative conditions, which derivesfrom the lack of transport of BDNF across the BBB. The lack of utilityof BDNF as a CNS therapeutic following peripheral administration isexpected and follows from the limiting role that is played by the BBB inthe development of neurotherapeutics, especially large molecule drugssuch as BDNF. BDNF does not cross the BBB, and the lack of transport ofthe neurotrophin across the BBB prevents the molecule from beingpharmacologically active in the brain following peripheraladministration. The lack of BDNF transport across the BBB means that theneurotrophin must be directly injected into the brain across the skullbone to be pharmacologically active in the CNS. However, when the BDNFis fused to a Trojan horse such as the HIR MAb, this neurotrophin is nowable to enter brain from blood following a non-invasive peripheral routeof administration such as intravenous intramuscular, subcutaneous,intraperitoneal, or even oral administration. Owing to the BBB transportproperties of this new class of molecule, it is not necessary toadminister the BDNF directly into the CNS with an invasive deliveryprocedure requiring penetration of the skull or spinal canal. Thereformulated fusion protein of the BDNF variant and the HIR MAb nowenables entry of BDNF into the brain from the blood, and the developmentof BDNF as a neurotherapeutic for human diseases.

As used herein, the term “BDNF” includes the pharmaceutically acceptablesalts and prodrugs, and prodrugs of the salts, polymorphs, hydrates,solvates, biologically-active fragments, biologically active variantsand stereoisomers of the naturally-occurring BDNF, as well as agonist,mimetic, and antagonist variants of the naturally-occurring BDNF andpolypeptide fusions thereof. Variants that include one or moredeletions, substitutions, or insertions in the natural sequence of theBDNF, in particular truncated versions of the native BDNF comprisingdeletion of one or more amino acids at the amino terminus, carboxylterminus, or both, are encompassed by the term “BDNF.” In someembodiments, the invention utilizes a carboxy-truncated variant of thenative BDNF, e.g., a variant in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore than 10 amino acids are absent from the carboxy-terminus of theBDNF. BDNF variants include the complete 119 amino acid BDNF, the 117 or118 amino acid variant with a truncated carboxyl terminus, variants witha truncated amino terminus, or variants with up to about a 20, 30, or40% change in amino acid composition, as long as the fusion proteinvariant still binds to the brain neuroprotection receptor with highaffinity. When an Ab, e.g., a MAb such as HIRMAb is used, additionalfusion protein variants can be produced with the substitution of aminoacids within either the framework region (FR) or the complementaritydetermining region (CDR) of either the light chain or the heavy chain ofthe Ab, e.g., HIRMAb, as long as the fusion protein binds with highaffinity to the endogenous receptor, e.g., HIR to promote transportacross the human BBB. Additional fusion protein variants can be producedby changing the composition or length of the linker peptide separatingthe fusion protein from the HIRMAb.

In some embodiments, the full-length 119 a.a. sequence of BDNF isutilized (SEQ ID NO: 39). In some embodiments, a one amino-acidcarboxy-truncated variant of BDNF is utilized (amino acids 1-118 of SEQID NO: 39). In some embodiments, a two amino-acid carboxy-truncatedvariant of BDNF is utilized (amino acids 1-117 of SEQ ID NO: 39). Insome embodiments, a three amino-acid carboxy-truncated variant of BDNFis utilized (amino acids 1-116 of SEQ ID NO: 39). In some embodiments, afour amino-acid carboxy-truncated variant of BDNF is utilized (aminoacids 1-115 of SEQ ID NO: 39). In some embodiments, a five amino-acidcarboxy-truncated variant of BDNF is utilized (amino acids 1-114 of SEQID NO: 39).

The sequence of human BDNF is given in SEQ ID NO: 39. In someembodiments, the invention utilizes a BDNF that is about 60, 70, 80, 90,95, 99, or 100% identical with the sequence of SEQ ID NO: 39, or atruncated version thereof, e.g., the 117 or 118 amino acid variant witha one- or two-amino acid truncated carboxyl terminus, or variants with atruncated amino terminus. In some embodiments, the invention utilizes atwo amino-acid carboxy-truncated 117 amino acid variant human BDNF witha sequence that is at least about 60, 70, 80, 90, 95, 99 or 100%identical to the sequence of amino acids 466-582 of SEQ ID NO: 24. Insome embodiments, the invention utilizes a two amino-acidcarboxy-truncated human 117 amino acid BDNF with a sequence thatincludes amino acids 466-582 of SEQ ID NO: 24.

Accordingly, BDNFs useful in the invention include peptides having atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 99%, or greater than 95% orgreater than 99% sequence identity, e.g., 100% sequence identity, to theamino acid sequences disclosed herein.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.). Thepercent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences])(100).

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of anotherpeptide. The FASTA algorithm is described by Pearson and Lipman, Proc.Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol.183:63 (1990). Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., SEQ ID NO:24 orSEQ ID NO: 39) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined forinula based uponthe length of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63 (1990).

The present invention also includes peptides having a conservative aminoacid change, compared with an amino acid sequence disclosed herein. Manysuch changes have been described specifically. More generally, forexample, variants can be obtained that contain one or more amino acidsubstitutions of SEQ ID NO:33, or truncated versions thereof, such asamino acids 466-582 of SEQ ID NO: 24. In these variants, e.g., an alkylamino acid is substituted for an alkyl amino acid in a BDNF peptideamino acid sequence, an aromatic amino acid is substituted for anaromatic amino acid in a BDNF peptide amino acid sequence, asulfur-containing amino acid is substituted for a sulfur-containingamino acid in a BDNF peptide amino acid sequence, a hydroxy-containingamino acid is substituted for a hydroxy-containing amino acid in a BDNFpeptide amino acid sequence, an acidic amino acid is substituted for anacidic amino acid in a BDNF peptide amino acid sequence, a basic aminoacid is substituted for a basic amino acid in BDNF peptide amino acidsequence, or a dibasic monocarboxylic amino acid is substituted for adibasic monocarboxylic amino acid in a BDNF peptide amino acid sequence.Among the common amino acids, for example, a “conservative amino acidsubstitution” is illustrated by a substitution among amino acids withineach of the following groups: (1) glycine, alanine, valine, leucine, andisoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine andthreonine, (4) aspartate and glutamate, (5) glutamine and asparagine,and (6) lysine, arginine and histidine. The BLOSUM62 table is an aminoacid substitution matrix derived from about 2,000 local multiplealignments of protein sequence segments, representing highly conservedregions of more than 500 groups of related proteins (Henikoff andHenikoff, Proc. Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, theBLOSUM62 substitution frequencies can be used to define conservativeamino acid substitutions that may be introduced into the amino acidsequences of the present invention. Although it is possible to designamino acid substitutions based solely upon chemical properties (asdiscussed above), the language “conservative amino acid substitution”preferably refers to a substitution represented by a BLOSUM62 value ofgreater than −1. For example, an amino acid substitution is conservativeif the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or3. According to this system, preferred conservative amino acidsubstitutions are characterized by a BLOSUM62 value of at least 1 (e.g.,1, 2 or 3), while more preferred conservative amino acid substitutionsare characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).

It also will be understood that amino acid sequences may includeadditional residues, such as additional N—or C-terminal amino acids, andyet still be essentially as set forth in one of the sequences disclosedherein, so long as the sequence retains sufficient biological proteinactivity to be functional in the compositions and methods of theinvention.

V. COMPOSITIONS

Compositions of the invention are useftil in one or more of: increasingserum half-life of a cationic compound, transporting an agent across theBBB, and/or retaining activity of the agent once transported across theBBB. Accordingly, in some embodiments, the invention providescompositions containing a neurotherapeutic agent covalently linked to astructure that is capable of crossing the blood brain barrier (BBB),where the composition is capable of producing an average elevation ofconcentration in the brain of the neurotherapeutic agent of at leastabout 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 ng/gram brain followingperipheral administration. The invention also provides compositionscontaining an agent that is covalently linked to a chimeric MAb to thehuman BBB insulin receptor. The invention further provides a fusionprotein containing a structure capable of crossing the BBB, covalentlylinked to a peptide that is active in the central nervous system (CNS),where the structure capable of crossing the blood brain barrier and thepeptide that is active in the central nervous system each retain anaverage of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of their activities, compared to their activities as separateentities. In certain embodiments, the invention further providescompositions that increase the serum half-life of cationic substances.The invention also provides pharmaceutical compositions that contain oneor more compositions of the invention and a pharmaceutically acceptableexcipient.

In some embodiments, the invention provides compositions containing aneurotherapeutic agent covalently linked to a structure that is capableof crossing the blood brain barrier (BBB), where the composition iscapable of producing an average elevation of concentration in the brainof the neurotherapeutic agent of at least about 1, 2, 3, 4, 5, 10, 20,30, 40, or 50 ng/gram brain following peripheral administration.

“Elevation” of the agent is an increase in the brain concentration ofthe agent compared to the concentration of the agent administered alone(i.e., not covalently linked to a structure that is capable of crossingthe BBB). In the case of agents for which only a small amount of theagent alone normally crosses the BBB, “elevation” may be an increase inthe agent compared to resting brain levels. “Average” refers to the meanof at least three, four, five, or more than five measurements,preferably in different individuals. The individual in which theelevation is measured is a mammal, such as a rat, or, preferably, aprimate, e.g., a monkey. An example of measurements of elevation of thelevel of a neurotherapeutic agent (BDNF) is given in Example 7.

In some embodiments, the structure that is capable of crossing the BBButilizes an endogenous BBB receptor mediated transport system, such as asystem that utilizes the insulin receptor, transferrin receptor, leptinreceptor, LDL receptor, or IGF receptor. In some embodiments, theendogenous BBB receptor mediated transport system is the insulin BBBreceptor mediated transport system. In some embodiments, the structurethat is capable of crossing the BBB is an antibody, e.g., a monoclonalantibody (MAb) such as a chimeric MAb. The antibody can be a chimericantibody with sufficient human sequence that it is suitable foradministration to a human. The antibody can be glycosylated ornonglycosylated; in some embodiments, the antibody is glycosylated,e.g., in a glycosylation pattern produced by its synthesis in a CHOcell. In embodiments in which the structure is an antibody, the covalentlinkage between the antibody and the neurotherapeutic agent may be alinkage between any suitable portion of the antibody and theneurotherapeutic agent, as long as it allows the antibody-agent fusionto cross the blood brain barrier and the neurotherapeutic agent toretain a therapeutically useful portion of its activity within the CNS.In certain embodiments, the covalent link is between one or more lightchains of the antibody and the neurotherapeutic agent. In the case of apeptide neurotherapeutic agent (e.g., a neurotrophin such as BDNF), thepeptide can be covalently linked by its carboxy or amino terminus to thecarboxy or amino terminus of the light chain (LC) or heavy chain (HC) ofthe antibody. Any suitable linkage may be used, e.g., carboxy terminusof light chain to amino terminus of peptide, carboxy terminus of heavychain to amino terminus of peptide, amino terminus of light chain toamino terminus of peptide, amino terminus of heavy chain to aminoterminus of peptide, carboxy terminus of light chain to carboxy terminusof peptide, carboxy terminus of heavy chain to carboxy terminus ofpeptide, amino terminus of light chain to carboxy terminus of peptide,or amino terminus of heavy chain to carboxy terminus of peptide. In someembodiments, the linkage is from the carboxy terminus of the HC to theamino terminus of the peptide. It will be appreciated that a linkagebetween terminal amino acids is not required, and any linkage whichmeets the requirements of the invention may be used; such linkagesbetween non-terminal amino acids of peptides are readily accomplished bythose of skill in the art.

In some embodiments, the invention utilizes BDNF, either the native formor truncated variants. Strikingly, it has been found that fusionproteins of these forms of BDNF retain full transport and activity. Thisis surprising because the neurotrophin is translated in vivo in cells asa prepro form and the prepro-BDNF is then converted into mature BDNFfollowing cleavage of the prepro peptide from the amino terminus of theBDNF. In order to preserve the prepro form of the BDNF, and thesubsequent cleavability of the prepro peptide, it would seem to benecessary to fuse the prepro BDNF to the amino terminus of either the HCor the LC of the targeting MAb. This could be inhibit the binding of theMAb for the target antigen, since the complementarity determiningregions (CDR) of the heavy chain or light chain of the MAb molecule,which comprise the antigen binding site of the MAb, are situated nearthe amino terminus of the heavy chain or light chains of the antibody.Therefore, fusion of the prepro-neurotrophin to the amino terminus ofthe antibody chains is expected to result in not only impairment ofantibody activity, but also an impairment of antibody folding followingtranslation. The present invention shows the unexpected finding that itis possible to fuse the mature form of a neurotrophin, such as a BDNFvariant (vBDNF), to the carboxyl terminus of the heavy chain of the HIRMAb. The production of this new genetically engineered fusion proteincreates a bi-functional molecule that binds with high affinity to boththe HIR and the trkB receptors.

In some embodiments, more than one molecule of the same neurotherapeuticagent is attached to the structure that crosses the BBB. For example, incompositions of the invention where a single neurotrophin is attached toan antibody, one molecule of the neurotrophin is attached to each heavychain, naturally producing a structure that is ideal for homodimerformation. This is the case for compositions containing BDNF.Neurotrophins such as BDNF require an obligatory formation of ahomo-dimeric structure to be biologically active, and to bind with highaffinity to the cognate receptor, e.g. TrkB. A naturally occurringhomo-dimeric structure between two BDNF molecules is formed when theneurotrophin is fused to a carboxyl terminus of the CH3 region of an IgGmolecule, as illustrated in FIG. 18. Without being bound by theory, itis thought that this may account for the unexpected finding ofessentially 100% of activity for the BDNF when bound to the IgG (see,e.g., FIG. 24).

In some embodiments, more than one type of neurotherapeutic agent can beattached to the structure that is capable of crossing the blood brainbarrier. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10 different neurotherapeutic agents may be attached to the structurethat is capable of crossing the blood brain barrier. In certainembodiments, 2 different neurotrophins are attached to an antibody to anendogenous BBB receptor-mediated transport system. Any combination ofneurotrophins may be used. Particularly useful in some embodiments ofthe invention are neurotrophins used as precursors for fusion proteinsthat cross the BBB are those that naturally form dimeric structures,similar to BDNF. Certain neurotrophins such as BDNF or NT-3 may formhetero-dimeric structures, and in some embodiments the inventionprovides a fusion protein constructed of one neurotrophin monomer fusedto one chain (e.g., heavy chain) of an antibody, e.g., of the HIRMAb,and another neurotrophin monomer fused to the second chain of theantibody. Typically, the molecular weight range of recombinant proteinsthat may be fused to the molecular Trojan horse ranges from 1000 Daltonsto 500,000 Daltons.

In some embodiments, more than one type of structure capable of crossingthe BBB, e.g., molecular Trojan horse, may be used. The differentstructures may be covalently attached to a single neurotherapeuticagent, e.g., a single neurotrophin such as BDNF, or multipleneurotherapeutics, e.g., multiple neurotrophins, or any combinationthereof. Thus, for example, in some embodiments either with the sameneurotrophin attached to each MTH or a different neurotrophin attached,or combinations of neurotrophins attached. Thus the neuroprotectiverecombinant protein can be fused to multiple molecular Trojan horsesthat undergo receptor-mediated transport across the blood-brain barrier,including monoclonal antibodies to the insulin receptor, transferrinreceptor, insulin-like growth factor (IGF) receptor, or the low densitylipoprotein (LDL) receptor or the endogenous ligand, including insulin,transferrin, the IGFs, or LDL. Ligands that traverse the blood-brainbarrier via absorptive-mediated transport may also be used as molecularTrojan horses including cationic proteins, or carbohydrate bearingproteins that bind to membrane lectins. The molecular weight range ofmolecular Trojan horses is 1000 Daltons to 500,000 Daltons.

The covalent linkage between the structure capable of crossing the BBBand the neurotherapeutic agent may be direct (e.g., a peptide bondbetween the terminal amino acid of one peptide and the terminal aminoacid of the other peptide to which it is linked) or indirect, via alinker. If a linker is used, it may be any suitable linker, e.g., apeptide linker. If a peptide linker is used, it may be 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10 amino acids in length. In some embodiments,a three amino acid linker is used. In some embodiments, the linker hasthe sequence ser-ser-met. The covalent linkage may be cleavable, howeverthis is not a requirement for activity of the system in someembodiments; indeed, an advantage of these embodiments of the presentinvention is that the fusion protein, without cleavage, is partially orfully active both for transport and for activity once across the BBB.

In some embodiments, a noncovalent attachment may be used. An example ofnoncovalent attachment of the MTH, e.g., MAb, to the large moleculetherapeutic neuroprotective factor is avidin/streptavidin-biotinattachment. Such an approach is further described in U.S. patentapplication Ser. No. 10/858,729, entitled “Anti-growth factor receptoravidin fusion proteins as universal vectors for drug delivery,” filedApr. 21, 2005, which is hereby incorporated by reference in itsentirety.

The neurotherapeutic agent may be any suitable neurotherapeutic agent,such as a neurotrophin. In some embodiments, the neurotherapeutic agentis a neurotrophin such as brain derived neurotrophic factor (BDNF),nerve growth factor (NGF), neurotrophin-4/5, fibroblast growth factor(FGF)-2 and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO),hepatocyte growth factor (HGF), epidermal growth factor (EGF),transforming growth factor (TGF)-α, TGF-β, vascular endothelial growthfactor (VEGF), interleukin-1 receptor antagonist (IL-1ra), ciliaryneurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF),neurturin, platelet-derived growth factor (PDGF), heregulin, neuregulin,artemin, persephin, interleukins, granulocyte-colony stimulating factor(CSF), granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF. TheBDNF may be native BDNF or a variant BDNF. Some embodiments utilize atwo amino acid carboxyl-truncated variant. The BDNF can be a human BDNF.In some embodiments, the BDNF contains a sequence that is about 60, 70,80, 90, 95, 99, or 100% identical to.the sequence of amino acids 466-582of SEQ ID NO: 24.

In some embodiments, the invention provides compositions containing aneurotherapeutic agent covalently linked to a structure that is capableof crossing the BBB where the composition is capable of producing anaverage elevation of concentration in the brain of the neurotherapeuticagent of at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, or 50 ng/grambrain following peripheral administration, where the neurotherapeuticagent is a neurotrophin and the structure that is capable of crossingthe BBB is a MAb to an endogenous BBB receptor mediated transportsystem. The antibody can be glycosylated or nonglycosylated; in someembodiments, the antibody is glycosylated, e.g., in a glycosylationpattern produced by its synthesis in a CHO cell. In certain embodiments,the neurotrophin is BDNF, e.g., a two amino acid carboxy-truncated BDNF.The MAb can be an antibody to the insulin BBB receptor mediatedtransport system, e.g., a chimeric MAb. The antibody can be a chimericantibody with sufficient human sequence that it is suitable foradministration to a human. In some embodiments, the insulin receptor isa human insulin receptor and the BDNF is a human BDNF. In someembodiments, the BDNF contains a sequence that is about 60, 70, 80, 90,95, 99, or 100% identical to the sequence of amino acids 466-582 of SEQID NO: 24. The BDNF can be covalently linked at its amino terminus tothe carboxy terminus of the heavy chain of the MAb, optionally with alinker between the termini, such as the three amino-acid linkerser-ser-met. In some embodiments, the heavy chain of the MAb contains asequence that is about 60, 70, 80, 90, 95, 99, or 100% identical toamino acids 20-462 of SEQ ID NO: 24. In some embodiments, the lightchain of the MAb contains a sequence that is about 60, 70, 80, 90, 95,99, or 100% identical to amino acids 21-234 of SEQ ID NO: 36.

In some embodiments, the invention provides compositions containing afusion MAb, where the fusion MAb is an antibody to the human insulin BBBreceptor mediated transport system linked to a two-amino acidcarboxy-truncated human BDNF. The BDNF is linked via its amino terminusto the carboxy terminus of the heavy chain of the antibody by aser-ser-met linker. The antibody is a chimeric antibody with sufficienthuman sequence that it is suitable for administration to a human. Insome embodiments, the invention provides compositions containing afusion MAb with a heavy chain-BDNF fusion, where the fusion MAb lightchain is at least about 60%, or about 70%, or about 80%, or about 90%,or about 95%, or about 99% identical to, or is substantially 100%identical to, amino acids 21-234 of SEQ ID NO: 36, and the heavychain-BDNF fusion is at least about 60%, or about 70%, or about 80%, orabout 90%, or about 95%, or about 99% identical to, or is substantially100% identical to amino acids 20-582 of SEQ ID NO: 24.

The invention also provides compositions containing an agent that iscovalently linked to a chimeric MAb to the human BBB insulin receptor.In some embodiments, the heavy chain of the MAb is covalently linked tothe agent to form a fusion protein. The agent can be any agent describedherein, i.e., any agent for which transport across the BBB is desired.In some embodiments, the agent is a therapeutic agent, such as aneurotherapeutic agent as described herein, e.g., a neurotrophin such asBDNF. In certain embodiments, the BDNF is a two amino acidcarboxyl-terminal truncated BDNF.

Strikingly, it has been found that multifunctional fusion proteins ofthe invention, e.g., difunctional fusion proteins, retain a highproportion of the activity of the separate portions, e.g., the portionthat is capable of crossing the BBB and the portion that is active inthe CNS. Accordingly, the invention further provides a fusion proteincontaining a structure capable of crossing the BBB, covalently linked toa peptide that is active in the central nervous system (CNS), where thestructure capable of crossing the blood brain barrier and the peptidethat is active in the central nervous system each retain an average ofat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% oftheir activities, compared to their activities as separate entities. Insome embodiments, the invention provides a fusion protein containing astructure capable of crossing the BBB, covalently linked to a peptidethat is active in the central nervous system (CNS), where the structurecapable of crossing the blood brain barrier and the peptide that isactive in the central nervous system each retain an average of at leastabout 50% of their activities, compared to their activities as separateentities. In some embodiments, the invention provides a fusion proteincontaining a structure capable of crossing the BBB, covalently linked toa peptide that is active in the central nervous system (CNS), where thestructure capable of crossing the blood brain barrier and the peptidethat is active in the central nervous system each retain an average ofat least about 60% of their activities, compared to their activities asseparate entities. In some embodiments, the invention provides a fusionprotein containing a structure capable of crossing the BBB, covalentlylinked to a peptide that is active in the central nervous system (CNS),where the structure capable of crossing the blood brain barrier and thepeptide that is active in the central nervous system each retain anaverage of at least about 70% of their activities, compared to theiractivities as separate entities. In some embodiments, the inventionprovides a fusion protein containing a structure capable of crossing theBBB, covalently linked to a peptide that is active in the centralnervous system (CNS), where the structure capable of crossing the bloodbrain barrier and the peptide that is active in the central nervoussystem each retain an average of at least about 80% of their activities,compared to their activities as separate entities. In some embodiments,the invention provides a fusion protein containing a structure capableof crossing the BBB, covalently linked to a peptide that is active inthe central nervous system (CNS), where the structure capable ofcrossing the blood brain barrier and the peptide that is active in thecentral nervous system each retain an average of at least about 90% oftheir activities, compared to their activities as separate entities. Insome embodiments, the structure capable of crossing the blood brainbarrier retains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95,99, or 100% of its activity, compared to its activity as a separateentity, and the peptide that is active in the central nervous systemretains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of its activity, compared to its activity as a separate entity.

As used herein, “activity” includes physiological activity (e.g.,ability to cross the BBB and/or therapeutic activity), and also bindingaffinity of the structures for their respective receptors.

Transport of the structure capable of crossing the BBB across the BBBmay be compared for the structure alone and for the structure as part ofa fusion structure of the invention by standard methods. For example,pharmacokinetics and brain uptake of the fusion structure, e.g., fusionprotein, by a model animal, e.g., a mammal such as a primate, may beused. Such techniques are illustrated in Example 7, which demonstratespharmacokinetics and brain uptake of a fusion protein of the inventionby the adult Rhesus monkey. Similarly, standard models for the functionof an agent, e.g. the therapeutic or protective function of atherapeutic agent, may also be used to compare the function of the agentalone and the function of the agent as part of a fusion structure of theinvention. See, e.g., Example 5, which demonstrates the activity of aneurotrophin alone and the same neurotrophin bound to a fusion proteinin a model system (hypoxia-reoxygenation in human neural cells). In bothExample 5 and Example 7, the fusion protein of the invention retainedabout 100% of the transport ability and the therapeutic function of itsindividual components, i.e., a structure capable of crossing the BBB (aMAb to the human insulin receptor) and a therapeutic agent (BDNF).

Alternatively, binding affinity for receptors may be used as a marker ofactivity. Binding affmity for the receptor is compared for the structurealone and for the structure when part of the fusion protein. A suitabletype of binding affmity assay is the competitive ligand binding assay(CLBA). For example, for fusion proteins containing MAbs to endogenousBBB receptor-mediated transport systems fused to a neurotrophin, a CLBAmay be used both to assay the affinity of the MAb for its receptor andthe neurotrophin for its receptor, either as part of the fusion proteinor as separate entities, and percentage affinity calculated. If, as insome embodiments, the peptide that is active in the CNS is highly ionic,e.g., cationic, causing a high degree of non-specific binding, suitablemeasures should be taken to eliminate the nonspecific binding. See,e.g., Example 4. “Average” measurements are the average of at leastthree separate measurements.

In embodiments of the above fusion proteins, the structure capable ofcrossing the blood brain barrier crosses the BBB on an endogenous BBBreceptor-mediated transporter, such as a transporter selected from thegroup consisting of the insulin transporter, the transferrintransporter, the leptin transporter, the LDL transporter, and the IGFreceptor. In some embodiments, the endogenous BBB receptor-mediatedtransporter is selected from the group consisting of the insulintransporter and the transferrin transporter. In some embodiments, theendogenous BBB receptor-mediated transporter is the insulin transporter,e.g., the human insulin transporter. The structure capable of crossingthe BBB can be an antibody, e.g., a MAb such as a chimeric MAb. Theantibody can be an antibody to an endogenous BBB receptor-mediatedtransporter, as described herein. The peptide that is active in the CNScan be a neurotherapeutic agent, e.g., a neurotrophin. In someembodiments, the neurotrophin is selected from the group consisting ofbrain-derived neurotrophic factor, nerve growth factor (NGF),neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs,neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor(HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF such asa truncated BDNF, e.g., a carboxyl-truncated BDNF. Thecarboxyl-truncated BDNF is lacking the two carboxyl terminal amino acidsin some embodiments. The structure capable of crossing the BBB and theneurotherapeutic agent are covalently linked by a peptide linker in someembodiments.

In certain embodiments, the invention provides compositions thatincrease the serum half-life of cationic substances. One limitation formany current therapeutics, especially cationic therapeutic peptides(e.g., BDNF) is their rapid clearance from the circulation. The positivecharge on the cationic substance, such as cationic peptides, rapidlyinteracts with negative charges on cell membranes, which triggers anabsorptive-mediated endocytosis into the cell, particularly liver andspleen. This is true not only for neurotherapeutics (where rapidclearance means only limited contact with the BBB and thus limitedability to cross the BBB) but for other agents as well, such as cationicimport peptides such as the tat peptide, or cationic proteins (e.g.protamine, polylysine, polyarginine) that bind nucleic acids, orcationic proteins such as avidin that bind biotinylated drugs.Surprisingly, fusion compositions of the invention that include acationic therapeutic peptide covalently linked to an immunoglobulin showgreatly enhanced serum half-life compared to the same peptide when itwas not covalently part of a fusion immunoglobulin. This is an importantfinding, because it shows that the fusion of a highly cationic protein,e.g., BDNF, to an immunoglobulin, e.g. HIRMAb, has two important andunexpected effects: I) it greatly enhances the serum half-life of thecationic protein, and 2) it does not accelerate the blood clearance ofthe immunoglobulin to which it is attached, e.g., the HIRMAb. Prior workshows that the noncovalent attachment of a cationic therapeutic peptide,e.g., the cationic BDNF to a monoclonal antibody greatly accelerated theblood clearance of the antibody, owing to the cationic nature of theBDNF, which greatly enhances hepatic uptake. The work in FIG. 27A andExample 7 shows that when the cationic therapeutic peptide, e.g., BDNFis re-engineered as an IgG fusion protein, the plasma pharmacokineticsis dominated by the IgG moiety, and that the blood level of the BDNFremains high for a prolonged period; indeed, the serum half-life of theBDNF in the fusion protein is at least about 100 times that of the BDNFalone.

Accordingly, in some embodiments, the invention provides compositioncomprising a cationic therapeutic peptide covalently linked to animmunoglobulin, wherein the cationic therapeutic peptide in thecomposition has a serum half-life that is an average of at least about1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,100, or more than about 100-fold greater than the serum half-life of thecationic therapeutic peptide alone. In some embodiments, the inventionprovides a composition comprising a cationic therapeutic peptidecovalently linked to an immunoglobulin, wherein the cationic therapeuticpeptide in the composition has a mean residence time (MRT) in the serumthat is an average of at least about 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than about 100-foldgreater than the serum half-life of the cationic therapeutic peptidealone. In some embodiments, the invention provides compositioncomprising a cationic therapeutic peptide covalently linked to animmunoglobulin, wherein the cationic therapeutic peptide in thecomposition has a systemic clearance rate that is an average of at leastabout 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80,90, 100, or more than about 100-fold slower than the systemic clearancerate of the cationic therapeutic peptide alone. In some embodiments, theinvention provides composition comprising a cationic therapeutic peptidecovalently linked to an immunoglobulin, wherein the cationic therapeuticpeptide in the composition has average blood level after peripheraladministration that is an average of at least about 1.5, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than about100-fold greater than the average blood level after peripheraladministration of the cationic therapeutic peptide alone.

In some embodiments, the cationic therapeutic peptide comprises aneurotherapeutic agent. Examples of neurotherapeutic agents that arecationic peptides interferons, interleukins, cytokines, or growthfactors with an isoelectric point (pI) above 8. In some embodiments, theneurotherapeutic agent is a neurotrophin. Cationic peptide neurotrophinsinclude BDNF, NT-3, NT4/5, NGF, and FGF-2. In some embodiments, theneurotrophin is BDNF.

In some embodiments, the immunoglobulin is an antibody to an endogenousBBB receptor-mediated transport system. In some embodiments, theendogenous BBB receptor-mediated transport system is selected from thegroup consisting of the insulin BBB transport system, the BBBtransferrin receptor, the BBB leptin receptor, the BBB IGF receptor, orthe BBB lipoprotein receptor. In some embodiments, the antibody is anantibody to the endogenous insulin BBB receptor-mediated transportsystem. Antibodies can be any suitable antibody as described herein.

Pharmaceutical compositions The invention also provides pharmaceuticalcompositions that contain one or more compositions of the invention anda pharmaceutically acceptable excipient. A thorough discussion ofpharmaceutically acceptable carriers/excipients can be found inRemington's Pharmaceutical Sciences, Gennaro, A R, ed., 20th edition,2000: Williams and Wilkins PA, USA. Pharmaceutical compostions of theinvention include compositions suitable for administration via anyperipheral route, including intravenous, subcutaneous, intrmuscular,intraperitoneal injection; oral, rectal, transbuccal, pulmonary,transdermal, intranasal, or any other suitable route of peripheraladministration.

The compostions of the invention are particular suited for injection,e.g., as a pharmaceutical composition for intravenous, subcutaneous,intramuscular, or intraperitonal administration. Aqueous compositions ofthe present invention comprise an effective amount of a composition ofthe present invention, which may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, e.g., a human,as appropriate. As used herein, “pharmaceutically acceptable carrier”includes any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic compositions is contemplated. Supplementary activeingredients can also be incorporated into the compositions.

Exemplary pharmaceutically acceptable carriers for injectablecompositions can include salts, for example, mineral acid salts such ashydrochlorides, hydrobromides, phosphates, sulfates, and the like; andthe salts of organic acids such as acetates, propionates, malonates,benzoates, and the like. For example, compositions of the invention maybe provided in liquid form, and formulated in saline based aqueoussolution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms. The prevention of the action of microorganisms can bebrought about by various antibacterial and antiftngal agents, forexample, parabens, chlorobutanol; phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate, and gelatin.

For human administration, preparations meet sterility, pyrogenicity,general safety, and purity standards as required by FDA and otherregulatory agency standards. The active compounds will generally beformulated for parenteral administration, e.g., formulated for injectionvia the intravenous, intramuscular, subcutaneous, intralesional, orintraperitoneal routes. The preparation of an aqueous composition thatcontains an active component or ingredient will be known to those ofskill in the art in light of the present disclosure. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use in preparing solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, methods ofpreparation include vacuum-drying and freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed

The term “unit dose” refers to physically discrete units suitable foruse in a subject, each unit containing a predetermined-quantity of thetherapeutic composition calculated to produce the desired responses,discussed above, in association with its administration, i.e., theappropriate route and treatment regimen. The quantity to beadministered, both according to number of treatments and unit dose,depends on the subject to be treated, the state of the subject and theprotection desired. The person responsible for administration will, inany event, determine the appropriate dose for the individual subject.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 1.0 to 100 milligrams or even about 0.01 to 1.0grams per dose or so. Multiple doses can also be administered. In someembodiments, a dosage of about 2.5 to about 25 mg of a fusion protein ofthe invention is used as a unit dose for administration to a human,e.g., about 2.5 to about 25 mg of a fusion protein of BDNF and a HIRMAb.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other alternativemethods of administration of the present invention may also be used,including but not limited to intradermal administration (See U.S. Pat.Nos. 5,997,501; 5,848,991; and 5,527,288), pulmonary administration (SeeU.S. Pat. Nos. 6,361,760; 6,060,069; and 6,041,775), buccaladministration (See U.S. Pat. Nos. 6,375,975; and 6,284,262),transdermal administration (See U.S. Pat. Nos. 6,348,210; and 6,322,808)and transmucosal administration (See U.S. Pat. No. 5,656,284). All suchmethods of administration are well known in the art. One may also useintranasal administration of the present invention, such as with nasalsolutions or sprays, aerosols or inhalants. Nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions. Thus, the aqueous nasal solutionsusually are isotonic and slightly buffered to maintain a pH of 5.5 to6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations and appropriate drug stabilizers, if required,may be included in the formulation. Various commercial nasalpreparations are known and include, for example, antibiotics andantihistamines and are used for asthma prophylaxis.

Additional formulations, which are suitable for other modes ofadministration, include suppositories and pessaries. A rectal pessary orsuppository may also be used. Suppositories are solid dosage forms ofvarious weights and shapes, usually medicated, for insertion into therectum or the urethra. After insertion, suppositories soften, melt ordissolve in the cavity fluids. For suppositories, traditional bindersand carriers generally include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in any suitable range, e.g., in the range of 0.5%to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations, or powders. Incertain defined embodiments, oral pharmaceutical compositions willcomprise an inert diluent or assimilable edible carrier, or they may beenclosed in a hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations can contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried, and may conveniently be between about 2 to about 75% of theweight of the unit, or between about 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, such as gum tragacanth, acacia, cornstarch, orgelatin; excipients, such as dicalcium phosphate; a disintegratingagent, such as corn starch, potato starch, alginic acid and the like; alubricant, such as magnesium stearate; and a sweetening agent, such assucrose, lactose or saccharin may be added or a flavoring agent, such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup of elixir may contain the active compoundssucrose as a sweetening agent, methylene and propyl parabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Insome embodiments, an oral pharmaceutical composition may be entericallycoated to protect the active ingredients from the environment of thestomach; enteric coating methods and formulations are well-known in theart.

VI. NUCLEIC ACIDS, VECTORS, CELLS, AND MANUFACTURE

The invention also provides nucleic acids, vectors, cells, and methodsof production.

A. Nucleic acids

In some embodiments, the invention provides nucleic acids that code forproteins or peptides of the invention. In certain embodiments, theinvention provides a single nucleic acid sequence containing a firstsequence coding for a light chain of an immunoglobulin and secondsequence coding a heavy chain of the immunoglobulin, where either thefirst sequence also codes for a peptide that is expressed as a fusionprotein of the peptide covalently linked to the light chain, or thesecond sequence also codes for a peptide that is expressed as a fusionprotein of the peptide covalently linked to the heavy chain. In someembodiments, the invention provides nucleic acid sequences, and in someembodiments the invention provides nucleic acid sequences that are atleast about 60, 70, 80, 90, 95, 99, or 100% identical to a particularnucleotide sequence. For example, in some embodiments, the inventionprovides a nucleic acid containing a first sequence that is at leastabout 60, 70, 80, 90, 95, 99, or 100% identical to nucleotides 58-1386of SEQ ID NO: 33 and a second sequence that is at least about 60, 70,80, 90, 95, 99, or 100% identical to nucleotides 1396-1746 of SEQ ID NO:33.

For sequence comparison, of two nucleic acids, typically one sequenceacts as a reference sequence, to which test sequences are compared. Whenusing a sequence comparison algorithm, test and reference sequences areentered into a computer, subsequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, including but not limited to, by thelocal homology algorithm of Smith and Waterman (1970) Adv. Appl. Math.2:482c, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=4 and a comparison of both strands. The BLAST algorithm is typicallyperformed with the “low complexity” filter turned off. The BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The invention provides nucleic acids that code for any of the peptidesof the invention. In some embodiments, the invention provides a singlenucleic acid sequence containing a gene coding for a light chain of animmunoglobulin and a gene coding for a fusion protein, where the fusionprotein includes a heavy chain of the immunoglobulin covalently linkedto a peptide. In some embodiments, the peptide is a therapeutic peptide.In some embodiments the peptide is a neurotherapeutic peptide, e.g., aneurotrophin such as BDNF. In some embodiments, the BDNF is a two aminoacid carboxy-truncated BDNF. In some embodiments, the immunoglobulin isan IgG. In some embodiments, the IgG is a MAb, such as a chimeric MAb.The antibody can be an antibody to a transport system, e.g., anendogenous BBB receptor-mediated transport system such as the endogenousBBB receptor-mediated insulin transport system. In some embodiments, theendogenous BBB receptor-mediated insulin transport system is a humanendogenous BBB receptor-mediated insulin transport system and whereinthe peptide to which the immunoglobulin heavy chain is covalently linkedis human BDNF. Any suitable peptide, neurotherapeutic peptide,neurotrophin, BDNF, antibody, monoclonal antibody, or chimeric antibody,as described herein, may be coded for by the nucleic acid, combined as afusion protein and coded for in a single nucleic acid sequence. As iswell-known in the art, owing to the degeneracy of the genetic code, anycombination of suitable codons may be used to code for the desiredfusion protein. In addition, other elements useful in recombinanttechnology, such as promoters, termination signals, and the like, mayalso be included in the nucleic acid sequence. Such elements arewell-known in the art. In addition, all nucleic acid sequences describedand claimed herein include the complement of the sequence.

In some embodiments that code for a BDNF, e.g., a variant BDNF, as acomponent of the fusion protein, the BDNF contains a sequence that isabout 60, 70,80, 90, 95, 99, or 100% identical to the sequence of aminoacids 466-582 of SEQ ID NO: 24. In some embodiments, the BDNF is linkedat its amino terminus to carboxy terminus of the heavy chain of theimmunoglobulin, e.g., MAb. The heavy chain of the MAb can comprise asequence that is about 60, 70, 80, 90, 95, 99 or 100% identical to aminoacids 20-462 of SEQ ID NO: 24. In some embodiments, the light chain ofthe immunoglobulin, e.g., MAb, comprises a sequence that is about 60,70, 80, 90, 95, 99 or 100% identical to amino acids 21-234 of SEQ ID NO:36. The nucleic acid can further contain a nucleic acid sequence thatcodes for a peptide linker between the heavy chain of the MAb and theBDNF. In some embodiments, the linker is S-S-M. The nucleic acid mayfurther contain a nucleic acid sequence coding for a signal peptide,wherein the signal peptide is linked to the heavy chain. Any suitablesignal peptide, as known in the art or subsequently developed, may beused. In some embodiments, the signal peptide attached to the heavychain comprises a sequence that is about 60, 70, 80, 90, 95, 99, or 100%identical to amino acids 1-19 of SEQ ID NO: 24. In some embodiments, thenucleic acid contains a nucleic acid sequence coding for another signalpeptide, wherein the other signal peptide is linked to the light chain.The signal peptide linked to the light chain can comprise a sequencethat is about 60, 70, 80, 90, 95, 99, or 100% identical to amino acids1-20 of SEQ ID NO: 36. The nucleic acid can contain a nucleic acidsequence coding for a selectable marker. In some embodiments theselectable marker is DHFR. The sequence of the DHFR can be about 60, 70,80, 90, 95, 99, or 100% identical to amino acids 1-187 of SEQ ID NO: 38.

In certain embodiments, the invention provides a nucleic acid comprisinga first sequence that codes for a neurotherapeutic peptide, e.g., aneurotrophin such as BDNF, in the same open reading frame as a secondsequence that codes for an immunoglobulin component. The immunoglobulincomponent can be, e.g., a light chain or a heavy chain, e.g., that is atleast about 60, 70, 80, 90, 95, 99, or 100% identical to nucleotides58-1386-of SEQ ID NO: 33 and a second sequence that is at least about60, 70, 80, 90, 95, 99, or 100% identical to nucleotides 1396-1746 ofSEQ ID NO: 33. In some embodiments, the nucleic acid also contains athird sequence that is at least about 60, 70, 80, 90, 95, 99, or 100%identical to nucleotides 61-702 of SEQ ID NO: 35. In some embodiments,the nucleic acid further contains a fourth sequence that codes for afirst signal peptide and a fifth sequence that codes for a second signalpeptide. In some embodiments, the fourth sequence is at least about 60,70, 80, 90, 95, 99, or 100% identical to nucleotides 1-57 of SEQ ID NO:33 and the fifth sequence is at least about 60, 70, 80, 90, 95, 99, or100% identical to nucleotides 1-60 of SEQ ID NO: 35. In someembodiments, the nucleic acid further contains a sequence that codes fora selectable marker, such as dihydrofolate reductase (DHFR). In someembodiments, the sequence that codes for the DHFR is at least about 60,70, 80, 90, 95, 99, or 100% identical to nucleotides 1-561 of SEQ ID NO:37.

B. Vectors

The invention also provides vectors. The vector can contain any of thenucleic acid sequences described herein. In some embodiments, theinvention provides a single tandem expression vector containing nucleicacid coding for an antibody heavy chain fused to a peptide, e.g., atherapeutic peptide such as a neurotrophin, and nucleic acid coding fora light chain of the antibody, all incorporated into a single piece ofnucleic acid, e.g., a single piece of DNA. The single tandem vector canalso include one or more selection and/or amplification genes. A methodof making an exemplary vector of the invention is provided in theExamples. However, any suitable techniques, as known in the art, may beused to construct the vector.

The use of a single tandem vector has several advantages over previoustechniques. The transfection of a eukaryotic cell line withimmunoglobulin G (IgG) genes generally involves the co-transfection ofthe cell line with separate plasmids encoding the heavy chain (HC) andthe light chain (LC) comprising the IgG. In the case of a IgG fusionprotein, the gene encoding the recombinant therapeutic protein may befused to either the HC or LC gene. However, this co-transfectionapproach makes it difficult to select a cell line that has equally highintegration of both the HC and LC-fusion genes, or the HC-fusion and LCgenes. The approach to manufacturing the fusion protein utilized incertain embodiments of the invention is the production of a cell linethat is permanently transfected with a single plasmid DNA that containsall the required genes on a single strand of DNA, including theHC-fusion protein gene, the LC gene, the selection gene, e.g. neo, andthe amplification gene, e.g. the dihydrofolate reductase gene. As shownin the diagram of the fusion protein tandem vector in FIG. 12, theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

C. Cells

The invention further provides cells that incorporate one or more of thevectors of the invention. The cell may be a prokaryotic cell or aeukaryotic cell. In some embodiments, the cell is a eukaryotic cell. Insome embodiments, the cell is a mouse myeloma hybridoma cell. In someembodiments, the cell is a Chinese hamster ovary (CHO) cell. Exemplarymethods for incorporation of the vector(s) into the cell are given inthe Examples. However, any suitable techniques, as known in the art, maybe used to incorporate the vector(s) into the cell. In some embodiments,the invention provides a cell capable of expressing an immunoglobulinfusion protein, where the cell is a cell into which has been permanentlyintroduced a single tandem expression vector, where both theimmunoglobulin light chain gene and the gene for the immunoglobulinheavy chain fused to the therapeutic agent, are incorporated into asingle piece of nucleic acid, e.g., DNA. In some embodiments, theinvention provides a cell capable of expressing an immunoglobulin fusionprotein, where the cell is a cell into which has been permanentlyintroduced a single tandem expression vector, where both theimmunoglobulin heavy chain gene and the gene for the immunoglobulinlight chain fused to the therapeutic agent, are incorporated into asingle piece of nucleic acid, e.g., DNA. The introduction of the tandemvector may be by, e.g., permanent integration into the chromsomalnucleic acid, or by, e.g., introduction of an episomal genetic element.

D. Methods of manufacture

In addition, the invention provides methods of manufacture. In someembodiments, the invention provides a method of manufacturing animmunoglobulin fusion protein, where the fusion protein contains animmunoglobulin heavy chain fused to a therapeutic agent, by permanentlyintroducing into a eukaryotic cell a single tandem expression vector,where both the immunoglobulin light chain gene and the gene for theimmunoglobulin heavy chain fused to the therapeutic agent, areincorporated into a single piece of nucleic acid, e.g., DNA. In someembodiments, the invention provides a method of manufacturing animmunoglobulin fusion protein, where the fusion protein contains animmunoglobulin light chain fused to a therapeutic agent, by permanentlyintroducing into a eukaryotic cell a single tandem expression vector,where both the immunoglobulin heavy chain gene and the gene for theimmunoglobulin light chain fused to the therapeutic agent, areincorporated into a single piece of nucleic acid, e.g., DNA. In someembodiments, the introduction of the vector is accomplished by permanentintegration into the host cell genome. In some embodiments, theintroduction of the vector is accomplished by introduction of anepisomal genetic element containing the vector into the host cell.Episomal genetic elements are well-known in the art In some embodiments,the therapeutic agent is a neurotherapeutic agent. In some embodiments,the single piece of nucleic acid further includes one or more genes forselectable markers. In some embodiments, the single piece of nucleicacid further includes one or more amplification genes. In someembodiments, the immunoglobulin is an IgG, e.g., a MAb such as achimeric MAb. The methods may further include expressing theimmunoglobulin fusion protein, and/or purifying the immunoglobulinfusion protein. Exemplary methods for manufacture, including expressionand purification, are given in the Examples.

However, any suitable techniques, as known in the art, may be used tomanufacture, optionally express, and purify the proteins. These includenon-recombinant techniques of protein synthesis, such as solid phasesynthesis, manual or automated, as first developed by Merrifield anddescribed by Stewart et al. in Solid Phase Peptide Synthesis (1984).Chemical synthesis joins the amino acids in the predetermined sequencestarting at the C-terminus. Basic solid phase methods require couplingthe C-terminal protected α-amino acid to a suitable insoluble resinsupport. Amino acids for synthesis require protection on the α-aminogroup to ensure proper peptide bond formation with the preceding residue(or resin support). Following completion of the condensation reaction atthe carboxyl end, the α-amino protecting group is removed to allow theaddition of the next residue. Several classes of a-protecting groupshave been described, see Stewart et al. in Solid Phase Peptide Synthesis(1984), with the acid labile, urethane-based tertiary-butyloxycarbonyl(Boc) being the historically preferred. Other protecting groups, and therelated chemical strategies, may be used, including the base labile9-fluorenylmethyloxycarbonyl (FMOC). Also, the reactive amino acidsidechain functional groups require blocking until the synthesis iscompleted. The complex array of functional blocking groups, along withstrategies and limitations to their use, have been reviewed by Bodanskyin Peptide Synthesis (1976) and, Stewart et al. in Solid Phase PeptideSynthesis (1984).

Solid phase synthesis is initiated by the coupling of the describedC-terminal α-protected amino acid residue. Coupling requires activatingagents, such as dicyclohexycarbodiimide (DCC) with or without1-hydroxybenzo-triazole (HOBT), diisopropylcarbodiimide (DIIPC), orethyldimethylaminopropylcarbodiimide (EDC). After coupling theC-terminal residue, the α-amino protected group is removed bytrifluoroacetic acid (25% or greater) in dichloromethane in the case ofacid labile tertiary-butyloxycarbonyl (Boc) groups. A neutralizing stepwith triethylamine (10%) in dichloro-methane recovers the free amine(versus the salt). After the C-terminal residue is added to the resin,the cycle of deprotection, neutralization and coupling, withintermediate wash steps, is repeated in order to extend the protectedpeptide chain. Each protected amino acid is introduced in excess (threeto five fold) with equimolar amounts of coupling reagent in suitablesolvent. Finally, after the completely blocked peptide is assembled onthe resin support, reagents are applied to cleave the peptide form theresin and to remove the side chain blocking groups. Anhydrous hydrogenfluoride (HF) cleaves the acid labile tertiary-butyloxycarbonyl (Boc)chemistry groups. Several nucleophilic scavengers, such asdimethylsulfide and anisole, are included to avoid side reactionsespecially on side chain functional groups.

VII. METHODS

The invention also provides methods. In some embodiments, the inventionprovides methods for transport of an agent active in the CNS across theBBB in an effective amount. In some embodiments, the invention providestherapeutic, diagnostic, or research methods. Diagnostic methods includethe development of peptide radiopharmaceuticals capable of transportacross the BBB, such as the fusion of a peptide ligand, orpeptidomimetic MAb for an endogenous receptor in the brain, followed bythe radiolabelling of the fusion protein, followed by systemicadministration, and external imaging of the localization within thebrain of the peptide radiopharmaceutical.

Neurotrophin drug development illustrates the problems encountered whendevelopment of the delivery of agents active in the CNS, e.g., CNS drugdevelopment, is undertaken in the absence of a parallel program indelivery across the BBB, e.g., CNS drug delivery. The advances in themolecular neurosciences during the Decade of the Brain of the 1990s ledto the cloning, expression and purification of more than 30 differentneurotrophic factors, including BDNF, nerve growth factor (NGF),neurotrophin-4/5, fibroblast growth factor (FGF)-2 and other FGFs,neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growth factor(HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). These natural substances are powerful restorativeagents in the brain and produce neuroprotection when the protein isinjected directly into the brain. In addition, the direct injection ofBDNF into the brain is a potent stimulant to new brain cell formationand neurogenesis.

Neurotrophins such as BDNF must be injected directly into the brain toachieve a therapeutic effect, because the neurotrophin does not crossthe BBB. Therefore, it is not expected that neurotrophic factors willhave beneficial effects on brain disorders following the peripheral(intravenous, subcutaneous) administration of these molecules. Duringthe 1990s, there were attempts to develop neurotrophic factors for thetreatment of a chronic neurodegenerative disorder, amyotrophic lateralsclerosis (ALS). The clinical protocols administered the neurotrophicfactor by subcutaneous administration, even though the neurotrophin mustpass the BBB to be therapeutic in neurodegenerative disease. Theclinical trials went forward and all neurotrophin phase III clinicaltrials for ALS failed. Subsequently, attempts were made to administerneurotrophins via intra-cerebroventricular (ICV) infusion, or convectionenhanced diffusion (CED), but these highly invasive modes of deliverywere either ineffective or toxic. Given the failure of neurotrophinmolecules, per se, as neurotherapeutics, more recent theories proposethe development of neurotrophin small molecule mimetics, neurotrophingene therapy, or neurotrophin stem cell therapy.

However, neurotherapeutics can be developed as drugs for peripheralroutes of administration, providing the neurotherapeutic is enabled tocross the BBB. Attachment of the neurotherapeutic, e.g. a neurotrophinsuch as BDNF to a MTH, e.g., the chimeric HIRMAb, offers a new approachto the non-invasive delivery of neurotherapeutics to the CNS in animals,e.g., mammals such as humans for the treatment of acute brain and spinalcord conditions, such as focal brain ischemia, global brain ischemia,and spinal cord injury, and chronic treatment of neurodegenerativedisease, including prion diseases, Alzheimer's disease (AD), Parkinson'sdisease (PD), Huntington's disease (HD), ALS, multiple sclerosis,transverse myelitis, motor neuron disease, Pick's disease, tuberoussclerosis, lysosomal storage disorders, Canavan's disease, Rett'ssyndrome, spinocerebellar ataxias, Friedreich's ataxia, optic atrophy,and retinal degeneration.

Accordingly, in some embodiments the invention provides methods oftransport of an agent active in the CNS from the peripheral circulationacross the BBB in an effective amount, where the agent is covalentlyattached to a structure that crosses the BBB, and where the agent aloneis not transported across the BBB in an effective amount. In someembodiments the invention provides methods of transport ofneurotherapeutic agent from the peripheral circulation across the BBB ina therapeutically effective amount, where the neurotherapeutic agent iscovalently attached to a structure that crosses the BBB, and where theneurotherapeutic agent alone is not transported across the BBB in atherapeutically effective amount.

The invention also provides, in some embodiments, methods of treatmentof disorders of the CNS by peripheral administration of an effectiveamount of a therapeutic agent, e.g., a neurotherapeutic agent covalentlylinked to a structure that is capable of crossing the BBB, where theagent alone is not capable of crossing the BBB in an effective amountwhen administered peripherally. In some embodiments, the CNS disorder isan acute disorder, and, in some cases, may require only a singleadministration of the agent. In some embodiments, the CNS disorder is achronic disorder and may require more than one administration of theagent.

In some embodiments, the effective amount, e.g., therapeuticallyeffective amount is such that a concentration in the brain is reached ofat least about 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 100, or more than 100 ng/gram brain. In someembodiments, a therapeutically effective amount, e.g., of a neurotrophinsuch as BDNF, is such that a brain level is achieved of about 0.1 to1000, or about 1-100, or about 5-50 ng/g brain. In some embodiments, theneurotherapeutic agent is a neurotrophin. In some embodiments, theneurotrophin is selected from the group consisting of BDNF, nerve growthfactor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2 andother FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), transforming growth factor(TGF)-α, TGF-β, vascular endothelial growth factor (VEGF), interleukin-1receptor antagonist (IL-1ra), ciliary neurotrophic factor (CNTF),glial-derived neurotrophic factor (GDNF), neurturin, platelet-derivedgrowth factor (PDGF), heregulin, neuregulin, artemin, persephin,interleukins, granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, or stemcell factor (SCF). In some embodiments, the neurotrophin is BDNF, e.g. atruncated BDNF, such as the carboxyl-truncated BDNFs described herein.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a neurotrophin, where theneurotrophin is capable of crossing the BBB to produce an averageelevation of neurotrophin concentration in the brain of at least about0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100,or more than 100 ng/gram brain following said peripheral administration,and where the neurotrophin remains at the elevated level for about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10 days after a singleadministration. In some embodiments, the neurotrophin remains at a levelof greater than about 1 ng/g brain, or about 2 ng/g brain, or about 5ng/g brain for about 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 daysafter a single administration. In some embodiments, the neurotrophin isBDNF, including truncated versions thereof.

In some embodiments, the invention provides methods of treating adisorder of the CNS by peripherally administering to an individual inneed of such treatment an effective amount of a composition of theinvention. The term “peripheral administration,” as used herein,includes any method of administration that is not direct administrationinto the CNS, i.e., that does not involve physical penetration ordisruption of the BBB. “Peripheral administration” includes, but is notlimited to, intravenous intramuscular, subcutaneous, intraperitoneal,intranasal, transbuccal, transdermal, rectal, transalveolar(inhalation), or oral administration. Any suitable composition of theinvention, as described herein, may be used. In some embodiments, thecomposition is a neurotrophin covalently linked to a chimeric HIR-MAb.In some embodiments, the neurotrophin is a BDNF. In some embodiments,the BDNF is a variant as described herein, such as a carboxyl-terminaltruncated variant.

A “disorder of the CNS” or “CNS disorder,” as those terms are usedherein, encompasses any condition that affects the brain and/or spinalcord and that leads to suboptimal function. In some embodiments, thedisorder is an acute disorder. Acute disorders of the CNS include focalbrain ischemia, global brain ischemia, brain trauma, spinal cord injury,acute infections, status epilepticus, migrane headache, acute psychosis,suicidal depression, and acute anxiety/phobia. In some embodiments, thedisorder is a chronic disorder. Chronic disorders of the CNS includechronic neurodegeneration, retinal degeneration, depression, chronicaffective disorders, lysosmal storage disorders, chronic infections ofthe brain, brain cancer, stroke rehabilitation, inborn errors ofmetabolism, autism, mental retardation. Chronic neurodegenerationincludes neurodegenerative diseases such as prion diseases, Alzheimer'sdisease (AD), Parkinson's disease (PD), Huntington's disease (HD),multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), transversemyelitis, motor neuron disease, Pick's disease, tuberous sclerosis,lysosomal storage disorders, Canavan's disease, Rett's syndrome,spinocerebellar ataxias, Friedreich's ataxia, optic atrophy, and retinaldegeneration, and aging of the CNS.

In some embodiments, the invention provides methods of treatment of theretina, or for treatment or prevention of blindness. The retina, likethe brain, is protected from the blood by the blood-retinal barrier(BRB). The insulin receptor is expressed on both the BBB and the BRB,and the HIRMAb has been shown to deliver therapeutics to the retina viaRMT across the BRB. BDNF is neuroprotective in retinal degeneration, butit was necessary to inject the neurotrophin directly into the eyeball,because BDNF does not cross the BRB. In some embodiments, fusionproteins of the invention are used to treat retinal degeneration andblindness with a route of administration no more invasive than anintravenous or subcutaneous injection, because the HIRMAb delivers theBDNF across the BRB, so that the neurotrophin is exposed to retinalneural cells from the blood compartment.

In some embodiments, the invention provides a method of treatment fordepression. A subset of patients with depression may have a braindeficiency of BDNF, and the correlation of single nucleotidepolymorphisms (SNPs) with affective disorders has been reported. Thedirect injection of BDNF into the brain has durable anti-depressanteffects in rodent model. The BDNF must be injected directly into thebrain, because the neurotrophin does not cross the BBB. In someembodiments, the invention provides a method for treating depression bychronic administration of a fusion protein of the invention, thuselevating the brain levels of BDNF and being therapeutic in thosepatients with depression and a reduced production of brain BDNF.

Formulations and administration. Any suitable formulation, route ofadministration, and dose of the compositions of the invention may beused. Formulations, doses, and routes of administration are determinedby those of ordinary skill in the art with no more than routineexperimentation. Compositions of the invention, e.g., fusion proteinsare typically administered in a single dose, e.g., an intravenous dose,of about 0.01-1000 mg, or about 0.05-500 mg, or about 0.1-100 mg, orabout 1-100 mg, or about 0.5-50 mg, or about 5-50 mg, or 5-50 mg, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 25, 40, 45,50, 60, 70, 80, 90, or 100 mg. Typically, for the treatment of acutebrain disease, such as stroke, cardiac arrest, spinal cord injury, orbrain trauma, higher doses may be used, whereas for the treatment ofchronic conditions such as Alzheimer's disease, Parkinson's disease,Huntington's disease, MS, ALS, transverse myelitis, motor neurondisease, Pick's disease, tuberous sclerosis, lysosomal storagedisorders, Canavan's disease, Rett's syndrome, spinocerebellar ataxias,Friedreich's ataxia, optic atrophy, and retinal degeneration, and aging,lower, chronic dosing may be used. Oral administration can require ahigher dosage than intravenous or subcutaneous dosing, depending on theefficiency of absorption and possible metabolism of the protein, as isknown in the art, and may be adjusted from the foregoing based onroutine experimentation.

For intravenous or subcutaneous administration, formulations of theinvention may be provided in liquid form, and formulated in saline basedaqueous solution of varying pH (5-8), with or without detergents suchpolysorbate-80 at 0.01-1%, or carbohydrate additives, such mannitol,sorbitol, or trehalose. Commonly used buffers include histidine,acetate, phosphate, or citrate.

Dosages for humans can be calculated from appropriate animal data. Forexample, human dosing of a BDNF-MAB conjugate is based on pre-clinicalpharmacokinetic studies, and these measurements have been performed in 2species, rats, and Rhesus monkeys. Prior work in 3 models of cerebralischemia in rats demonstrated the range of effective doses of theBDNF-MAb conjugate is 5-50 ug/rat or 20-200 ug/kg of BDNF in the form ofthe BDNF-MAb conjugate. Since the BDNF component of the fusion proteinmolecule is 16%, and the HIRMAb component is 84%, the dose of fusionprotein is 6-fold greater than the equivalent BDNF dose. Pharmacokineticstudies in rats show these doses produce a concentration of the BDNF inthe form of conjugate in plasma of 50-500 ng/mL, and in brain of 5-50ng/g. Pharmacokinetic studies in adult Rhesus monkeys with the HIRMAbshow that the average plasma concentration in the first hour is 0.1%injected dose (ID)/mL, and that the brain concentration is 0.02% ID/g.The brain concentration of the fusion protein is about 0.01% ID/g (FIG.27). Owing to the scaling effect between species, and to the 10-foldlarger body size and brain size of humans relative to Rhesus monkeys,the projected plasma and brain concentrations in humans are 0.01% ID/mLand 0.001% ID/g respectively. Since the human brain is 1200 grams,then >1% of the injected dose is delivered to the human brain, which isa level of brain uptake comparable to small molecules. Given an injecteddose of fusion protein of 2.5-25 mg in humans, the expected 60 minplasma concentration is 250-2500 ng/ml of fusion protein, and theexpected 60 min brain concentration is 25-250 ng/g of fusion protein,which is equivalent to 440 ng/gram brain of BDNF. The 5 mg and 25 mgfusion protein doses in humans will produce a brain concentration of theBDNF that is neuroprotective in either global or regional brainischemia. Since the BDNF comprises 16% of the fusion protein, theeffective doses of BDNF administered to humans is 0.4 or 4.0 mg,respectively, for the 2.5 or 25 mg dose of fusion protein.

The fusion protein may also be formulated for chronic use for thetreatment of a chronic CNS disorder, e.g., neurodegenerative disease,stroke or brain/spinal cord injury rehabilitation, or depression.Chronic treatment may involve daily, weekly, bi-weekly administration ofthe composition of the invention, e.g., fusion protein eitherintravenously, intra-muscularly, or subcutaneous in formulations similarto that used for acute treatment. Alternatively, the composition, e.g.,fusion protein may be formulated as part of a bio-degradable polymer,and administered on a monthly schedule.

Combination therapies. The composition of the invention, e.g., fusionprotein may be administered as part of a combination therapy. Thecombination therapy involves the administration of a composition of theinvention in combination with another therapy for the CNS disorder beingtreated. If the composition of the invention is used in combination withanother CNS disorder method or composition, any combination of thecomposition of the invention and the additional method or compositionmay be used. Thus, for example, if use of a composition of the inventionis in combination with another CNS disorder treatment agent, the two maybe administered simultaneously, consecutively, in overlapping durations,in similar, the same, or different frequencies, etc. In some cases acomposition will be used that contains a composition of the invention incombination with one or more other CNS disorder treatment agents.

Other CNS disorder treatment agents that may be used in methods of theinvention include, without limitation, thromolytic therapy for stroke,amyloid-directed therapy for Alzheimers disease, dopamine restorationtherapy for Parkinsons disease, RNA interference therapy for geneticdisorders, cancer, or infections, and anti-convulsant therapy forepilepsy. Dosages, routes of administration, administration regimes, andthe like for these agents are well-known in the art.

In some embodiments, the composition, e.g., fusion protein isco-administered to the patient with another medication, either withinthe same formulation or as a separate composition. For example, thefusion protein could be formulated with another fusion protein that isalso designed to deliver across the human blood-brain barrier arecombinant protein other than BDNF. The fusion protein may beformulated in combination with other large or small molecules.

VIII. KITS

Compositions of the invention, e.g., fusion proteins, may be provided asa kit that includes the formulation, e.g., fusion protein in a containerand in suitable packaging. The composition can be provided in a drypowder form, in solid form (i.e., lyophilized), in solution, or insuspension. If the composition is a protein, to the proteins may havebeen added emulsifiers, salts, preservatives, other proteins, nucleicacids, protease inhibitors, antibiotics, perfumes, polysaccharides,adhesive agents, polymers, microfibrils, oils, etc. The composition ispackaged for transport, storage and/or use by a consumer. Such packagingof therapeutic compositions for transport, storage, and use iswell-known in the art. Packaged compositions may include furthercomponents for the dispensing and storage of the composition, and mayalso include separately packaged diluent comprised of, e.g., sterilewater or a suitable buffer, for solubilizing the formulation, e.g.,fusion protein prior to administration to the patient. Kits of theinvention may also include written materials, including instructions foruse, results of clinical studies, desired outcome and expected course oftreatment, information about precautions and side effects, and the like.The kits may optionally further contain other components, such asgloves, scissors, tape, implements for disposal of used vials and otherwaste, masks, antiseptic, antibiotics, and the like.

EXAMPLES Example 1

Construction of the Single Tandem Vector Containing Complete Genes ForIgG-Neurotherapeutic Fusion

Genetic engineering of a eukaryotic expression vector encoding the heavychain (HC) of the fusion protein is outlined in FIG. 1. The final fusionprotein HC expression vector was designated pHIRMAb-BDNF, or clone 416.This vector was designed to produce a fusion protein, comprised of aBDNF variant fused to the HC of the HIRMAb. Either BDNF or a variant ofBDNF (vBDNF) can be fused to the HIRMAb. The vBDNF differs from nativehuman BDNF by substitution of certain amino acids, such as a vBDNF wherethe 2 amino acids at the carboxyl terminus of BDNF are absent in vBDNF.The clone 416 plasmid was derived from clone 400, which produces the HCof the chimeric form of the HIRMAb, and a cDNA encoding mature humanvBDNF, which was produced as described in FIG. 2. Clone 400 encodes achimeric human IgG1 that is derived from a chromosomal fragment encodingthe human IgG1 constant region, and is comprised of both intron and exonsequences. The HC gene of the chimeric HIRMAb in clone 400 was subclonedat the BamHI site of the pCR II plasmid to facilitate engineering of thestop codon located at the 3′-end of the CH3 region by site directedmutagenesis (SDM). The engineering of the stop codon located at the endof the CH3 region was performed by site-directed mutagenesis to producea SspI site. The SspI site allows for insertion of the vBDNF cDNA (FIG.3) by blunt-end ligation into clone 400 to form clone 415. SDM wasperformed using the QuickChange SDM kit (Stratagene, Calif.). Sense andcomplementary mutagenic primers were designed in a way that the CH3 stopcodon (aaTGAg) is mutated to SspI site (aaTATt). In addition, primerscontained 15 nucleotides of the stop codon 5′- and 3′-surroundingregion; the sequence of these primers, designated SDM-SspI forward (FWD)and reverse (REV) are given in Table 1. TABLE 1 Nucleotide sequence ofoligodeoxynucleotides used for engineering plasmid clone 416SDM-SspI-FWD (SEQ ID NO.1) CCTGTCTCCGGGTAAATATTTGCGACGGCCGGCAAGSDM-SspI-REV (SEQ ID NO.2) CTTGCCGGCCGTCGCAAATATTTACCCGGAGACAGGXhoI-NheI linker FWD (SEQ ID NO.3)ATGCTCGAGGAATTCCCATGGATGATGGCTAGCAAGCTTATG XhoI-NheI linker REV (SEQ IDNO.4) CATAAGCTTGCTAGCCATCATCCATGGGAATTCCTCGAGCATXhoI-NheI (underlined) is a Universal linker that contains the followingRE sites: XhoI-EcoRI-NcoI-NheI-HindIII. SDM = site-directed mutagenesis;FWD = forward; REV = reverse

DNA sequence analysis of the IgG promoter region revealed the presenceof additional SspI sites in this region. Therefore, it was firstnecessary to release the HC promoter region (PRO-VH) by digestion ofclone 404 with XhoI and NheI, and the clone 404 was re-closed with aXhoI-NheI linker which produced clone 405 (-Pro-VH). The sequence of theforward and reverse ODNs used to produce the XhoI-NheI. linker are givenin Table 1. Plasmid clone 405 (-Pro-VH) now carries the single SspI siteintroduced by SDM. The human vBDNF cDNA was subcloned at SspI to form anintermediate plasmid named clone 414 (not shown). The complete fusionprotein HC expression cassette was then reconstructed by subcloning ofthe PRO-VH fragment previously deleted to form clone 415. The fusionprotein HC gene was then subcloned in the eukaryotic expression vector,clone 400, at the BamHI site to form clone 416.

The vBDNF cDNA was produced by PCR via either of 2 equivalentapproaches. In one approach, a prokaryotic expression plasmid, pHTBS01,isolated as an expressed sequence tag (EST), and encoding human BDNF,was digested with BamHI and BpII, and gel purified, and re-ligated withT4 ligase and the 5′-end linker to produce clone 412 (FIG. 2). Thesequence of the forward and reverse ODNs used to produce the 5′-endlinker are given in Table 2. TABLE 2 Engineering of 5′- and 3′-endlinkers of vBDNF cDNA 1) 5′-end linker of vBDNF FWD-ODN (SEQ ID NO.5)TCCGGATCCTCGCGAGTATGCACTCTGACCCTGCCCGTCGAGGTGAGCTG AGCGTG 2) 5′-endlinker of vBDNF REV-ODN (SEQ ID NO.6)CACGCTCAGCTCACCTCGACGGGCAGGGTCAGAGTGCATACTCGCGAGGA TCCGGA 3) 3′-endlinker of vBDNF FWD-ODN (SEQ ID NO.7)AGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAG GTGATCGCGACTCGAGATG4) 3′-end linker of vBDNF REV-ODN (SEQ ID NO.8)CATCTCGAGTCGCGATCACCTTTTAATGGTCAATGTACATACACAAGAAGTGTCTATCCTTATGAATCGCCAGCCAATTCTCTTTTTGCTATCCATGGTA AGGGCCCGCACGTACGACT5) vBDNF-PCR-U87 FWD-ODN (SEQ ID NO.9) ATCTCGCGAGTATGCACTCTGACCCTGCC 6)vBDNF-PCR-U87 REV-ODN (SEQ ID NO.10) ATCTCGCGATCACCTTTTAATGGTCAA

-   -   SEQ ID NO 5 and 6: Artificial forward (FWD) and reverse (REV)        oligodeoxynucleotide (ODN) duplex linkers were designed to        engineer a mature vBDNF cDNA that allows for insertion into the        CH3 open reading frame (orf) of clone 400 heavy chain (HC) to        form clone 416 (FIG. 1). The 5′-end linker is flanked by BamHI        and EspI, respectively, and it reconstructs the amino terminus        of the mature vBDNF. BamHI and EspI allow for directional        subcloning into the vBDNF intermediate plasmid clone 413 (FIG.        2). A Nrul site follows BamHI and it enables insertion of the        vBDNF into the HC vector (clone 405, FIG. 1) at the SspI site.        In addition, the linker also has “GT” immediately after NruI to        maintain the orf of the CH3 (FIG. 1). This modification        introduces a Ser-Ser-Met linker between CH3 and the vBDNF amino        terminus.    -   SEQ ID NO 7 and 8: The 3′-end linker contains SpII and XhoI to        reconstruct the COOH terminus of the mature vBDNF and introduces        a stop codon “TGA”. This linker has SpII, XhoI and NruI sites        for directional subcloning and insertion into clone 405 (FIGS. 1        and 2).    -   SEQ ID NO 9 and 10: FWD ODN reconstructs the amino terminus of        the mature vBDNF and introduces a Ser-Ser-Met linker. Nrul site        for insertion into the expression vector is underlined. REV ODN        introduces the TGA stop codon. NruI site for insertion into the        expression vector is underlined.

Clone 412 was then digested with XhoI and BsiWI, and gel purified, andre-ligated with T4 ligase and the 3′end linker to produce clone 413(FIG. 2). The sequence of the forward and reverse ODNs used to producethe 3′-end linker are given in Table 2. The vBDNF cDNA, encoding thevBDNF with a reconstructed stop codon, was released from clone 413 byNrul, and gel purified; the ethidium bromide stain of the agarose gel isshown in FIG. 3A. This gel shows the expected size of the vBDNF cDNA,0.4 kb, and the vector backbone, 3.5 kb. Alternatively, the BDNF cDNAwas produced by PCR from cDNA derived by reverse transcription ofpolyA+RNA isolated from human U87 glioma cells, which produceneurotrophins. The primers used to produce the vBDNF by PCR from theU87-derived cDNA are given in Table 2. This PCR produced the expected0.4 kb vBDNF cDNA (FIG. 3B). The 0.4 kb vBDNF fragment was then digestedwith NruI, and subcloned into clone 415, as described in FIG. 1, toproduce the full fusion protein HC expression cassette, which wasreleased by BamHI and subcloned into the original eukaryotic expressionplasmid to produce clone 416 (FIG. 1), the final expression plasmid forthe fusion protein HC. Clone 416 was analyzed by double digestion withNheI and BamHI and compared with that of the original clone 400, whichlacks the vBDNF. The agarose gel-separated products are shown in FIG.3C, where lanes 1 and 3 show the fragments generated from clone 416 andclone 400, respectively. Both plasmids produce a 6 kb vector backbone(upper of 3 bands in lanes 1 and 3), and a 2.5 kb promoter region (lowerof 3 bands in lanes 1 and 3). However, the size of the middle band is0.4 kb larger for clone 416, as compared to clone 400 (middle band,lanes 1 and 3). A negative clone is shown in lane 2 of FIG. 3C.

The nucleotide and amino acid sequence of the reconstructed carboxylterminus at the CH3 region of the HIRMAb HC, a 3-amino acid linker(Ser-Ser-Met), the vBDNF sequence, followed by a stop codon is shown inFIG. 4. The entire 2711 nucleotides (nt) comprising the fusion proteinHC gene of clone 416 is shown in FIG. 5. The ATG initiation codon andthe TGA stop codon are underlined. The human IgG1 constant region intronand exon sequences are shown in italics and bold font, respectively, inFIG. 5. The vBDNF nt sequence in the clone 416 vector is underlined inFIG. 5. These data show that intronic sequence is found between CH 1 andthe hinge region, between the hinge region and CH2, and between CH2 andCH3 regions of the human IgG1 constant region. The open reading frame(orf) of the fusion protein HC gene encodes for a 563 amino acidprotein, following cleavage of a 19 amino acid signal peptide, and theamino acid sequence of the fusion protein HC is shown in FIG. 6. Thesignal peptide is underlined; the cysteine (C) residues within theconstant region that form inter- or intra-chain disulfide bridges areshown in bold font; the serine-serine-methionine (SSM) linker betweenthe CH3 region of the IgG and the vBDNF is underlined; the singleN-linked glycosylation site, at the asparagine residue within CH2 inshown by bold underlined font (FIG. 6). The amino acid sequences of theindividual domains of the fusion protein HC protein are given in FIG. 7.The vBDNF domain of the fusion protein is comprised of 117 amino acids.

Clone 416 plasmid DNA was electroporated into mouse myeloma cells thathad previously been transfected with an expression plasmid encoding thelight chain (LC) of the chimeric HIRMAb. Since the vBDNF is fused onlyto the HC, there is no modification of the LC of the chimeric HIRMAb.Following selection of transfected cell lines, media from 96-well plateswere screened with an ELISA comprised of 2 anti-human IgG antibodies;one antibody is directed against the heavy chain of human IgG1, and theother antibody is directed against human kappa light chains. Myelomaclones encoding for intact fusion protein were isolated, and propagatedin a 10 L bioreactor. However, the production levels of the fusionprotein were low. This low production was attributed to several factors,including (i) transfection of the myeloma line by 3 separate expressionplasmids encoding the heavy chain gene, the light chain gene, and theantibiotic resistance gene; and (ii) the use of genomic fragment of theheavy and light chain genes with large intronic sequences. Therefore,the fusion protein expression plasmid was re-engineered with thefollowing features:

-   (1) the polymerase chain reaction (PCR) was used to convert genomic    fragments of the fusion protein HC and LC genes into ‘intron-less’    cDNA forms of the 2 genes-   (2) the cDNA forms the fusion protein HC and LC genes were placed on    a single ‘tandem vector’ in which the 2 genes were placed in    separate and tandem expression cassettes with separate promoters-   (3) the promoter driving the expression of the fusion protein HC and    LC genes was changed from the human IgG promoters to the    cytomegalovirus (CMV) promoter, to enable transfection of    non-myeloma cells, such as Chinese hamster ovary (CHO) cells-   (4) the tandem vector encoding fusion protein contains a gene    encoding for the dihydrofolate reductase (DHFR) gene, under a    separate SV40 promoter, to allow for methotrexate (MTX) selection of    CHO lines which contain amplification of the genome in the region of    the insertion of the expression vector.

In order to produce the fusion protein tandem vector, it was firstnecessary to produce intermediate plasmids, which separately encode cDNAforms of the fusion protein HC and LC genes. Eukaryotic expressionplasmids carrying the CMV promoter and the bovine growth hormone (BGH)poly-A (pA) transcription termination sequences, and designated pCD,were digested with NheI and XhoI and re-ligated with T4 ligase and anNheI-EcoRV-KpnI-ScaI-BamHI-XhoI linker, as shown in FIG. 8. The sequenceof the forward and reverse ODNs used to produce this linker are given inTable 3. TABLE 3 Nucleotide sequence of ODNs used for engineering ofintronless expression vectors 1) Linker NheI-EcoRV-KpnI-XcaI-BamHI-XhoIFWD ODN (SEQ ID NO.11) ATGGCTAGCGATATCGGTACCGTATACGGATCCCTCGAGATG 2)Linker NheI-EcoRV-KpnI-XcaI-BamHI-XhoI REV ODN (SEQ ID NO.12)CATCTCGAGGGATCCGTATACGGTACCGATATCGCTAGCCAT 3) PGR cloning of LC FWD ODNprimer (SEQ ID NO.13) GTGACAAACACAGACATAGGATATC 4) PCR cloning of LC REVODN primer (SEQ ID NO.14) ATGCTCGAGCTAACACTCTCCCCT 5) PCR cloning offusion protein HG FWD ODN primer (SEQ ID NO.15)ATGAATATTCCACCATGGAATGCAGC 6) PCR cloning of fusion protein HG REV ODNprimer (SEQ ID NO.16) ATAGGATCCTCACCTTTTAATGGTCAARE cloning sites are underlined: GATATC: EcoRV, CTCGAG: XhoI, AATATT:SspI, GGATCC: BamHI.

The resulting plasmid, designated pCD-linker (FIG. 8) was digested withEcoRV and BamHII and reclosed with T4 ligase and the fusion protein HCcDNA generated by PCR. For the PCR reaction, the above mentioned myelomaline that had been dual transfected with genomic constructs of thefusion protein HC (clone 416) and LC genes were digested and myelomaderived polyA+RNA was produced (part A in FIG. 8). Oligodeoxythymidine(ODT) primers were used to produced myeloma cDNA with reversetranscriptase from 0.5 ug of myeloma polyA+RNA, followed by a finalRNase digestion. From this cDNA, PCR was used to produce the cDNA formof the fusion protein HC gene, using the forward and reverse primersshown in Table 3, and high fidelity Pfu DNA polymerase. Similarly, thefusion protein LC cDNA was produced by PCR from the myeloma derivedcDNA, and the sequences of the forward and reverse PCR primers used toamplify the fusion protein LC cDNA are given in Table 3. Following PCR,the cDNA was applied to an 0.8% agarose gel, and all amplificationsyielded a single product, a 1.8 kb fusion protein HC cDNA (lane 1, FIG.3D), and a 0.7 kb fusion protein LC cDNA (lane 2, FIG. 3D). The fusionprotein HC PCR product was digested with SspI and BamHI and subclonedinto CD-linker to produce the clone 422a (FIG. 8), which is anintronless eukaryotic expression plasmid encoding the fusion protein HCcDNA. Clone 422a was analyzed by restriction endonuclease using NheI;digestion with this enzyme, which has a site in the new multiple cloningregion of the pCD vector, produced the expected 0.4 kb fragmentcorresponding to the fusion protein heavy chain variable region (VH)cDNA (lanes 14, FIG. 3E). The nucleotide sequence of the fusion proteinHC cDNA encoded by clone 422a is shown in FIG. 9A, which shows theintron sequences present in clone 416 (FIG. 5) have been deleted by thePCR of processed myeloma RNA. The amino acid sequence encoded by thefusion protein HC cDNA is given in FIG. 9B, and this amino acid sequenceis identical to that produced by the genomic fragment in clone 416 (FIG.6).

The fusion protein LC PCR product was digested with EcoRV and XhoI andsubcloned into CD-linker to produce the clone 423a (FIG. 10), which isan intronless eukaryotic expression plasmid encoding the fusion proteinLC cDNA. Clone 423a was analyzed by restriction endonuclease using EcoRVand BamHI; digestion with these enzymes, which have a site in the newmultiple cloning region of the pCD vector, produced the expected 0.7 kbfragment corresponding to the fusion protein LC cDNA (lanes 1-5, FIG.3F). The nucleotide sequence of the fusion protein LC cDNA encoded byclone 423a is shown in FIG. 11A, which shows the intron sequences havebeen deleted by the PCR of processed myeloma RNA. The amino acidsequence encoded by the fusion protein LC cDNA is shown in FIG. 11B.

Clones 422a and 423a were the precursors to the fusion protein tandemvector, as outlined in FIG. 12. In 2 steps, clone 422a was subjected toSDM to introduce an EcoRI site at the 3′-end of the fusion protein HCexpression cassette; the sequences of the forward and reverse SDMprimers are given in Table 4. TABLE 4 Nucleotide sequences of ODNs usedfor engineering of TV-12 1) EcoRI-SDM FWD ODN (SEQ ID NO.17)AAAAGGCCAGGAACCGAATTCAGATCTCGTTGCTGGCGTTTT 2) EcoRI-SDM REV ODN (SEQ IDNO.18) AAAACGCCAGCAACGAGATCTGAATTCGGTTCCTGGCCTTTT 3) EcoRI linker FWD(SEQ ID NO.19) ATCGAATTCAAGCTTGCGGCCGCGTATACAGATCTATC 4) EcoRI linkerREV (SEQ ID NO.20) GATAGATCTGTATACGCGGCCGCAAGCTTGAATTCGATEcoRI site in EcoRI-SDM ODN is underlined.The EcoRI linker introduces EcoRI-HindIII-NotI-XcaI RE sites.

In step 2, the mutated clone 422a was digested with EcoRI, blunt-ended,and re-ligated with the EcoRI-HindIII-NotI-XcaI linker to produce clone422a-I (FIG. 12). The sequence of the ODNs used to produce this EcoRIlinker are given in Table 4. Clone 422a-I was digested with EcoRI andHindIII, and closed with T4 ligase in the presence of the fusion proteinLC expression cassette to produce clone 422a-II (FIG. 12). The fusionprotein LC expression cassette was generated by digestion of clonepBS-LC-1 with EcoRI and HindIII. Clone pBS-LC-1 was produced fromEcoRV-digested pBS (Bluescript), T4 ligase, and the fusion protein LCexpression cassette produced by digestion of clone 423a with SspI (FIG.12). In parallel, a mouse DHFR expression cassette, containing the SV40promoter and the hepatitis C virus polyA region, was produced from thepFR400 plasmid (designated pDHFR) by digestion of the plasmid with SmaIand SaII (FIG. 12). The final fusion protein tandem vector was producedby subcloning the DHFR expression cassette into XcaI digested clone422a-II followed by closure with T4 ligase (FIG. 12). The fusion proteintandem vector was analyzed by restriction endonuclease, and the 11 kbplasmid was linearized by PvuI (lane 1, FIG. 3G). The 1.8 kb fusionprotein LC and 1.5 kb DHFR expression cassettes, and the 8 kb vectorbackbone including the fusion protein HC expression cassette werereleased by digestion with EcoRI and HindII (lane 2, FIG. 3G). Thetandem vector was subjected to DNA sequencing in both directions, andthe nucleotide sequence, and the deduced amino acid sequence of thefusion protein HC, the fusion protein LC, and the DHFR genes are shownin FIGS. 14, 15, and 16, respectively. The calculated MW of the fusionprotein HC and LC are 62,220 and 25,760 Da, respectively, not accountingfor any carbohydrate content of the fusion protein HC.

Example 2

Electroporation of CHO Cells With Fusion Protein Tandem Vector andCultivation in a Bioreactor.

The fusion protein tandem vector (FIG. 12) was linearized with PvuI andelectroporated into CHO-K1 cells followed by selection with G418 (375ug/ml) for 3 weeks. Positive clones were detected in 96 well plates witha human IgG ELISA that uses 2 primary antibodies to both the human IgG1HC and the human kappa LC. Cell lines of high copy number of thetransgene were selected by graded increases in MTX to 600 nM. TheMTX-selected cell line was grown in T175 flasks and then transferred toa 20L bioreactor with a 10 L volume of CHO cell serum free medium (SFM).As shown in FIG. 17, the CHO cells were maintained at high density inexcess of 10 million viable cells/mL for nearly 50 days in perfusionmode in the bioreactor. The secretion by these cells of the fusionprotein was detected by ELISA using antibodies to either human IgG or tohuman BDNF. As shown in FIG. 18, the fusion protein is a 1:1 fusion ofthe vBDNF to the carboxyl terminus of the HIRMAb heavy chain, whichresults in formation of the fusion protein heavy chain. This heavy chaincomplexes with the light chain, as shown in FIG. 18. Therefore, thefusion protein should react equally well to 3 antibodies directedagainst: (i) the human IgG1 HC, (ii) the human kappa LC; or (iii) humanBDNF. As shown in FIG. 19, there is a direct correlation in measurementof the fusion protein in the CHO cell medium depending on whetheranti-human IgG or anti-human BDNF antibodies are used in the ELISA.These ELISA results were confirmed with immunocytochemistry (ICC), whichshowed the CHO cells transfected with TV-120 were immunoreactive withantibodies to either human IgG or to human BDNF, and that the BDNFimmune signal was eliminated by absorption of the anti-BDNF antibodywith recombinant BDNF.

Example 3

Purification and Characterization of Bioreactor Produced Fusion Protein.

The conditioned medium obtained from the bioreactor under perfusion modewas passed through a 1 μm filter, and the medium collected in a 200 LBioprocess container under sterile conditions, which were maintained at4° C. in a glass door refrigerator contiguous with the bioreactor. Then,200 L batches of conditioned medium were passed through 1 μm and 0.4 μmpre-filters for the removal of cell debris. The medium was thenconcentrated with tangential flow filtration (TFF). The TFF system was aPellicon 2 model from Millipore and was comprised of five 0.5 m²filtration cassettes with a 30 kDa molecular weight cutoff and a totalsurface area of 2.5 m². A transmembrane gradient of 15 PSI was produced,which results in a reduction in volume of the 200 L to 2 L within 2hours. The concentrated medium was passed through an 0.22μ filter priorto elution through 100 mL Prosep A (Millipore) recombinant protein Aaffinity column. Following application of the sample, the column waswashed with buffer A (0.025 M NaCl, 0.025 M Tris, pH=7.4, 3 mM EDTA).The elution of CHO cell host protein (CHOP) was monitored at A280 with aShimadzu detector. The fusion protein was eluted with 0.1 M citric acid(pH=3) in tubes containing Tris base to cause immediate neutralizationto pH 7. The neutralized acid eluate pool was diluted with doubledistilled water until the conductivity was <7 mS, and the material wasapplied to a 50 mL Sepharose SP cation exchange column (Amersham) thathas been equilibrated with a 0.02 M Tris, pH=7.5. Following washing inthe Tris buffer, the residual CHOP was separated from the fusion proteinwith a linear NaCl gradient from 0 to 1 M NaCl. The fusion protein peakwas pooled and buffer exchanged and concentrated with a Milliporediafiltration unit with a 30 kDa molecular weight cutoff. The finalconcentrated antibody solution was sterile filtered (0.22 μm) and storedat 4° C. The fusion protein was purified to homogeneity on sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), asdemonstrated in FIG. 20. The size of the fusion protein heavy chain was68 kDa as compared to the size of the HIRMAb heavy chain, which was 54kDa. The difference between the size of the fusion protein and HIRMAbheavy chains reflects the added vBDNF monomer (14 kDa) fused to eachheavy chain of the fusion protein. The fusion protein reacts with bothanti-human IgG antibodies and anti-human BDNF antibodies on Westernblotting with the expected molecular weight size of the immunoreactivebands (FIG. 21). Isoelectric focusing (IEF) shows the isoelectric point(pI) of recombinant BDNF was highly cationic with a pI>10 (FIG. 22). Theobserved pI of the fusion protein was 8.5, and approximates the pI ofthe HIRMAb (FIG. 22). The observed pI of the fusion protein, 8.5, wasconsistent with the calculated pI, which is 9.04 and 5.27 for the fusionprotein HC and LC, respectively (http://scansite.mit.edu/).

Example 4

The Fusion protein is Bi-Functional and Binds With High Affinity to Boththe Human Insulin Receptor and to the Human trkB Receptor.

The affinity of the fusion protein for the HIR extracellular domain(ECD) was determined with a competitive ligand binding assay (CLBA)using the lectin affinity purified HIR ECD. CHO cells permanentlytransfected with the HIR ECD were grown in serum free media (SFM), andthe HIR ECD was purified with a wheat germ agglutinin affinity column.The HIR ECD was plated on Nunc-Maxisorb 96 well dishes and the bindingof the murine HIRMAb to the HIR ECD was detected by radioactivitymeasurements following addition of [¹²⁵I] murine HIRMAb as the ligand inthe binding assay (FIG. 23A). The binding of the [¹²⁵I] murine HIRMAb tothe HIR ECD was displaced by the addition of unlabeled fusion protein orHIRMAb as demonstrated in FIG. 23B. The CLBA shows comparable binding ofthe HIRMAb or the fusion protein. A Scatchard analysis using a highaffinity and low affinity binding site model and nonlinear regressionanalysis was performed to determine the affinity constant of the fusionprotein binding to the HIR. Both the fusion protein and the HIRMAb bindequally well to the HIR with a high affinity binding constant,Ki=0.63±0.07 nM (FIG. 23B).

The TrkB CLBA was designed for measurement of the affinity of the fusionprotein for recombinant human TrkB ECD. The design of a TrkB CLBA wasmade difficult by the cationic nature of BDNF, which causes a highdegree of nonspecific binding in the assay and this reduces thesensitivity of the assay. The nonspecific binding of BDNF could beeliminated by conjugation of 2000 Da polyethyleneglycol (PEG) to theprotein. A bifunctional PEG molecule, biotin-PEG²⁰⁰⁰-hydrazide (Hz), wascommercially obtained, and conjugated to BDNF to produceBDNF-PEG²⁰⁰⁰-biotin, as outlined in FIG. 24A; this molecule was used asthe “tracer” in the CLBA. The TrkB ECD was absorbed to ELISA plates andbinding of BDNF-PEG²⁰⁰⁰-biotin to the TrkB was detected colorimetricallywith avidin and biotin peroxidase (FIG. 24A). Prior studies showed theELISA signal (A490) was directly proportional to the amount of TrkBadded to the well. In addition, the assay had a very low blank and theA490 was <0.04 when no TrkB is plated. The binding of theBDNF-PEG²⁰⁰⁰-biotin to the TrkB was competitively displaced by therecombinant BDNF (FIG. 24B) or the fusion protein (FIG. 24C). TheScatchard analysis of the binding data using nonlinear regressionanalysis allowed for the computation of the Ki of binding of either BDNFor fusion protein to TrkB, as shown in FIG. 24B and 24C, respectively.The affinity of the fusion protein for TrkB was not statisticallydifferent from the affinity of the recombinant BDNF (FIG. 19B,C). Thenonspecific binding (NSB) of the assay was comparable for either BDNF orthe fusion protein. The NSB likely represents nonlinear cooperativebinding of the neurotrophin to the TrkB extracellular domain. The TrkBCLBA results shown in FIG. 24 indicate the affinity of fusion proteinfor the TrkB receptor was not changed following fusion of the vBDNF tothe carboxyl terminus of the HIRMAb heavy chain.

Neurotrophins such as BDNF require an obligatory formation of ahomo-dimeric structure to be biologically active, and to bind with highaffinity to the cognate receptor, e.g. TrkB. A naturally occurringhomo-dimeric structure between two BDNF molecules was formed when theneurotrophin was fused to a carboxyl terminus of the CH3 region of anIgG molecule, as illustrated in FIG. 18. The surprising observation ofthe maintenance of the high affinity binding of BDNF for TrkB (FIG. 24),despite fusion to the HIRMAb heavy chain (FIG. 18), is consistent withthe fact that BDNF normally binds to TrkB as a dimer.

Example 5

Human Neural Cells Subjected to Hypoxia are Neuroprotected by the FusionProtein With Equal Activity as Recombinant BDNF.

Human SH-SY5Y neural cells were exposed to 10 uM retinoic acid for 7days, which induces gene expression of trkB, the BDNF receptor. Thecells were then exposed to 16 hours of oxygen deprivation in a sealedchamber, with oxygen sensor. Excitotoxic neural damage was then inducedby 4 hours of re-oxygenation (FIG. 25A). During this 4 hourre-oxygenation period, the cells were exposed to either no treatment orequi-molar concentrations of human recombinant BDNF or fusion protein.As shown in FIG. 25B, the fusion protein was equipotent with nativehuman BDNF with respect to inducing neuroprotection in human neuralcells exposed to excitoxic ischemia-re-oxygenation.

Example 6

High Affinity Binding of Fusion Protein to Human Blood-Brain BarrierInsulin Receptor in Isolated Human Brain Capillaries.

Isolated human brain capillaries are used as an in vitro model system ofthe human BBB (FIG. 26A). The fusion protein was radiolabeled with3H—N-succinimidyl propionate, and added to the human brain capillariesto establish a radio-receptor assay (RRA) of fusion protein binding tothe HIR of the human BBB. [³H]-fusion protein is specifically bound tothe BBB, as the binding is self-inhibited by unlabeled fusion protein(FIG. 26B). The fusion protein is bound by the insulin receptor of thehuman BBB, because the murine HIRMAb (mHIRMAb) also inhibits binding of[³H]-fusion protein to the human BBB. The binding data in FIG. 26B werefit to a Scatchard plot with a non-linear regression analysis to producethe binding constants: K_(D)=0.55±0.07 nM, B_(max)=1.35±0.10pmol/mg_(p), and NSB=0.39±0.02 pmol/mg_(p), where K_(D) is thedissociation constant, Bmax is the maximal binding, and NSB is thenon-saturable binding. The KD is <1 nM, which indicate the fusionprotein binds the HIR on the human BBB with very high affinity.

Example 7

Pharmacokinetics and Brain Uptake of Fusion Protein by the Adult RhesusMonkey.

The fusion protein was tritiated with [³H]—N-succinimidyl propionate toa specific activity of 2.0 μCi/μg. A 5 year old female Rhesus monkey,weighing 5.2 kg, was administered by a single intravenous injection adose of 746 μCi (373 μg), and serum was collected at multiple timepoints over a 180 min period. The serum glucose of the anesthetized,overnight-fasted primate was constant throughout the 180 min studyperiod, and averaged 72±2 mg %, which indicates that the administrationof the HIRMAb based fusion protein caused no interference of theendogenous insulin receptor, and had no effect on glycemia control.

The serum removed from the anesthetized Rhesus monkey was analyzed fortotal radioactivity (FIG. 27A), and radioactivity that was precipitableby trichloroacetic acid (TCA) (FIG. 27B). At 180 minutes after druginjection, the animal was euthanized, and brain radioactivity wasanalyzed with the capillary depletion method (FIG. 27C), similar toprior work on the brain uptake of [¹²⁵I]-labeled murine HIRMAb in theRhesus monkey. Based on the specific activity of the [³H]-fusionprotein, the brain radioactivity was converted to ng per gram (g) brain,as shown in FIG. 27D, and this level was compared to the reportedendogenous concentration of BDNF in the adult primate brain.

The plasma pharmacokinetics analysis (FIG. 27A) shows that the fusionprotein of the genetically engineered HIRMAb and the BDNF is removedfrom blood at the same rate as the original murine HIRMAb. This is animportant finding, because it shows that the fusion of BDNF, a highlycationic protein, to the HIRMb does not accelerate the blood clearanceof the HIRMAb. Prior work shows that the attachment of the cationic BDNFto a monoclonal antibody greatly accelerates the blood clearance of theantibody, owing to the cationic nature of the BDNF, which greatlyenhances hepatic uptake. The work in FIG. 27A shows that when thecationic BDNF was re-engineered as an IgG fusion protein, the plasmapharmacokinetics was dominated by the IgG moiety, and that the bloodlevel of the BDNF remains high for a prolonged period.

The data in FIG. 27B show that when BDNF was re-formulated as an IgGfusion protein, the metabolic stability of the neurotrophin in blood wasgreatly enhanced, as compared to the native BDNF. Owing to its cationicnature, the native BDNF was rapidly removed from blood, and was rapidlydegraded into TCA-soluble radioactive metabolites (FIG. 27B). However,the TCA-insoluble form of the labeled fusion protein remains high duringthe 3 hours after an intravenous injection in the primate (FIG. 27B).The data in FIGS. 27A,B show the advantages of re-engineering aneurotrophin pharmaceutical as a fusion protein. The native neurotrophinwas rapidly removed from blood and was rapidly degraded. However, theplasma pharmacokinetics profile, and metabolic stability profile, of theneurotrophin resemble those of an IgG molecule, when theIgG-neurotrophin fusion protein was produced.

Native BDNF is not transported across the BBB. Similarly, a [³H]-mouseIgG2a isotype control antibody was not transported across the BBB in theadult Rhesus monkey, as the brain volume of distribution (VD) of the IgGat 180 minutes after an intravenous injection was equal to the plasmavolume, 18 μL/g (FIG. 27C, open bars). Conversely, the brain V_(D) ofthe [³H]-fusion protein exceeds 140 μl/g brain (FIG. 27C, closed bars).Capillary depletion analysis separates the brain vasculature from thepost-vascular supernatant, and allows detection of the transport of adrug through the BBB and into brain, as opposed to simple sequestrationof the drug by the brain vasculature. The brain VD of the post-vascularsupernatant of the [³H]-fusion protein was equal to the V_(D) of thebrain homogenate (FIG. 27C), which indicates the fusion protein wastransported through the BBB and into brain parenchyma.

The brain V_(D) of the fusion protein was converted into ng fusionprotein per gram brain, based on the specific activity of the[³H]-fusion protein, and this allowed for calculation of the total massof fusion protein in the brain, 24±1 ng/g, as shown in FIG. 27D. Thisvalue is >10-fold higher than the endogenous brain concentration of BDNFin the adult primate (45). Therefore, the administration of a dose of373 μg to a 5.2 kg Rhesus monkey, which is equal to a normalized dose of72 μg/kg of fusion protein, results in a marked increase in the brainconcentration of BDNF. Such an increase in brain BDNF, followingintravenous administration, is not possible with native BDNF, becausethe native BDNF does not cross the BBB. However, when BDNF isre-engineered in the form of the fusion protein, then pharmacologicallyactive levels of the neurotrophin in brain are achieved (FIG. 27D).

The data shows that: (1) the plasma mean residence time (MRT) of thefusion protein, 312 minutes, was 100-fold greater than the MRT fornative BDNF, which was 3.0 minutes, and (2) the systemic clearance ofthe fusion protein, 0.94 mL/min/kg, was 39-fold slower than the systemicclearance of the BDNF, which was 37 mL/min/kg. In other words, theaverage blood level of the recombinant protein was up to 100-foldgreater when the recombinant protein was re-formulated as an IgG fusionprotein. Thus, fusion of the BDNF to the molecular Trojan horse had 2benefits: (1) the molecular Trojan horse carried the BDNF across theblood-brain barrier (BBB), whereas the BDNF alone cannot cross the BBB,and (2) the molecular Trojan horse prevented the rapid loss from bloodof the neurotrophin; BDNF by itself lasts only about 3 minutes in theblood. Both of these properties serve to enhance the pharmacologicaleffect of the BDNF in brain following administration into the bloodstream. See, e.g., Table 5. TABLE 5 Pharmacokinetic parameters for[³H]-fusion protein and [³H]-BDNF Parameter [³H]-fusion protein[³H]-BDNF A₁ (% ID/ml) 0.147 ± 0.020 5.28 ± 0.60 A₂ (% ID/ml) 0.061 ±0.005 2.26 ± 0.32 k₁ (min⁻¹) 0.195 ± 0.050 1.75 ± 0.26 k₂ (hr⁻¹) 0.186 ±0.042 15.6 ± 0.6  t_(1/2) ¹ (min) 3.5 ± 0.9 0.42 ± 0.07 t_(1/2) ² (hr)3.7 ± 0.9 0.045 ± 0.001 CL_(SS) (ml/min/kg) 0.94 ± 0.16 37.0 ± 2.5  MRT(min) 312 ± 78  3.0 ± 0.3A_(1,) A_(2,) k_(1,) and k₂ are the intercepts and slopes of thebi-exponential function describing the decay in plasma concentrationwith time. The parameters for the fusion protein were determined for theRhesus monkey, and the parameters for BDNF were determined in the adultrat. All data are normalized for differences in body weight.t_(1/2) ¹ and t_(1/2) ² are computed from k₁ and k₂, respectively, andare the half-times of the decay curves for each exponent.CL_(ss) and MRT are the steady state clearance and mean residence time,respectively, and are computed from A_(1,) A_(2,) k_(1,) and k₂ usingstandard pharmacokinetic formulations.

Example 8

Neuroprotection in Regional Brain Ischemia by Conjugates of BDNF and aBBB Molecular Trojan Horse.

Numerous attempts have been made to develop neuroprotective agents forthe treatment of acute stroke. There have been no successes to datebecause the neuroprotective drugs are either too toxic, in the case ofcertain small molecules, or ineffective, because the drug does not crossthe BBB. BDNF is neuroprotective when injected directly in the brain inparallel with experimental stroke in rodents and regional brainischemia. The BDNF must be injected across the skull bone into thebrain, because this large molecule drug does not cross the BBB. Sincethe BBB is intact in the early hours after regional brain ischemia, andsince BDNF does not cross the BBB, then there is no neuroprotectiveeffect in the ischemic brain following the intravenous administration ofBDNF alone. To deliver BDNF across the BBB, the neurotrophin wasattached to a mouse MAb to the rat transferrin receptor (TfR). Thispeptidomimetic MAb carries BDNF across the BBB, and the combinedBDNF-MAb conjugate is highly neuroprotective following delayedintravenous administration in experimental stroke, because the BDNF isable to cross the BBB and enter the brain from blood. Once inside thebrain, and behind the BBB, the BDNF activates its cognate receptor,trkB, which then induces neuroprotection in ischemic neurons, and stopsthe apoptotic death cycle. The neuroprotective effect of the BDNF-MAbconjugate demonstrates a dose response effect, a time response effect,and is long-lasting, as the neuroprotection at 7 days is identical tothe neuroprotection at 1 day after a single intravenous administrationof the BDNF-MAb conjugate. See, e.g., Zhang and Pardridge (2001) BrainRes. 889:49-56, and Zhang and Pardridge (2001) Stroke 32:1378-1374,which are incorporated by reference herein in their entirety. The fusionprotein would also be neuroprotective in human stroke, since the BDNF isfused to an MAb to the HIR, which rapidly binds to both the human BBB invitro, and is rapidly transported across the primate BBB in vivo.

Example 9

Neuroprotection in Global Brain Ischemia of Conjugates of BDNF and a BBBMolecular Trojan Horse.

The direct injection of BDNF into the brain is also neuroprotective intransient forebrain ischemia (TFI), such as might occur after a cardiacarrest. However, intravenous BDNF is not neuroprotective in TFI, becausethe BDNF does not cross the BBB, and because the BBB is intact in theearly hours after TFI, when neuroprotection is still possible.Conversely, intravenous BDNF was neuroprotective in TFI if the BDNF wasattached to a mouse MAb against the rat transferrin receptor (TfR),which acts as a molecular Trojan horse to ferry the BDNF across the BBBand into brain. Adult rats were subjected to TFI, which resulted in aflat-line electroencephalogram (EEG) for approximately a 10-minuteperiod. The animals were resuscitated and then administered 1 of 4different therapeutics intravenously: (a) buffer, (b) unconjugated BDNF,(c) the receptor specific MAb without the BDNF attached, and (d) theBDNF-MAb conjugate. In the case of the animals treated with saline,unconjugated BDNF, or MAb alone, there was no neuroprotection ofpyramidal neurons in the CA1 sector of hippocampus. However, in the caseof the BDNF-MAb conjugate, there is complete normalization of CA1pyramidal neuron density following delayed intravenous administration.See, e.g., Wu and Pardridge (199), PNAS (USA) 96:254-259, which isincorporated by reference herein in its entirety. This shows that BDNFis strongly neuroprotective in global brain ischemia following delayedintravenous administration, providing the BDNF is attached to a BBBmolecular Trojan horse. The recombinant fusion protein of BDNF and areceptor specific MAb could be given following cardiac arrest to preventpermanent brain damage.

Example 10

BDNF is Neuroprotective in Brain and Spinal Cord Injury if theNeurotrophin Can Access Brain Cells.

BDNF is neuroprotective in brain injury, providing the neurotrophin isinjected directly through the skull bone, because BDNF does not crossthe BBB. BDNF is also neuroprotective in brain subjected to excitotoxicinjury by neurotoxins, and is neuroprotective in brain infected with thehuman immune deficiency virus (HIV)-1. BDNF is also neuroprotective inacute spinal cord injury; however, the BDNF must be administered bydirect infusion into the spinal canal, because the BDNF does not crossthe blood-spinal cord barrier, which is the same as the BBB in theforebrain. In all these cases, the intravenous administration of BDNFwould not be neuroprotective, because the BDNF does not cross the BBB,and the BBB is intact in brain injury in the early hours after theinjury, when neuroprotection is still possible. Conversely, the BDNFfusion protein would be neuroprotective in these conditions followingintravenous administration, because the BDNF is fused to the BBBmolecular Trojan horse, and is able to penetrate the brain and spinalcord from the blood following peripheral administration.

Example 11

BDNF is Neuroprotective in Chronic Neurodegenerative Conditions of Brainif the Neurotrophin Can Access Brain Cells.

Neurotrophins, such as BDNF can be developed as drugs for peripheralroutes of administration, providing the neurotrophin is enabled to crossthe BBB. Fusion of BDNF to the chimeric HIRMAb offers a new approach tothe non-invasive delivery of BDNF to the brain in humans for the chronictreatment of neurodegenerative disease, including prion diseases,Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease(HD), ALS, transverse myelitis, motor neuron disease, Pick's disease,tuberous sclerosis, lysosomal storage disorders, Canavan's disease,Rett's syndrome, spinocerebellar ataxias, Friedreich's ataxia, opticatrophy, and retinal degeneration, and brain aging.

Example 12

BDNF as a Therapeutic in Retinal Degeneration and Blindness.

The retina, like the brain, is protected from the blood by theblood-retinal barrier (BRB). The insulin receptor is expressed on boththe BBB and the BRB, and the HIRMAb has been shown to delivertherapeutics to the retina via RMT across the BRB (Zhang et al, (2003)Mol. Ther. 7: 11-18). BDNF is neuroprotective in retinal degeneration,but it was necessary to inject the neurotrophin directly into theeyeball, because BDNF does not cross the BRB. The fusion protein couldbe used to treat retinal degeneration and blindness with a route ofadministration no more invasive than an intravenous or subcutaneousinjection, because the HIRMAb would deliver the BDNF across the BRB, sothat the neurotrophin would be exposed to retinal neural cells from theblood compartment.

Example 13

BDNF as a Therapeutic for Depression.

A subset of patients with depression may have a brain deficiency ofBDNF, and the correlation of single nucleotide polymorphisms (SNPs) withaffective disorders has been reported. The direct injection of BDNF intothe brain has durable anti-depressant effects in rodent model. The BDNFmust be injected directly into the brain, because the neurotrophin doesnot cross the BBB. The chronic administration of the fusion proteinwould provide a means for elevating the brain levels of BDNF, and may betherapeutic in those patients with depression and a reduced productionof brain BDNF.

Example 14

Method of Manufacturing IgG Fusion Proteins.

The transfection of a eukaryotic cell line with immunoglobulin G (IgG)genes generally involves the co-transfection of the cell line withseparate plasmids encoding the heavy chain (HC) and the light chain (LC)comprising the IgG. In the case of a IgG fusion protein, the geneencoding the recombinant therapeutic protein may be fused to either theHC or LC gene. However, this co-transfection approach makes it difficultto select a cell line that has equally high integration of both the HCand LC-fusion genes, or the HC-fusion and LC genes. The preferredapproach to manufacturing the fusion protein is the production of a cellline that is permanently transfected with a single plasmid DNA thatcontains all the required genes on a single strand of DNA, including theHC-fusion protein gene, the LC gene, the selection gene, e.g. neo, andthe amplification gene, e.g. the dihydrofolate reductase gene. As shownin the diagram of the fusion protein tandem vector in FIG. 12, theHC-fusion gene, the LC gene, the neo gene, and the DHFR gene are allunder the control of separate, but tandem promoters and separate buttandem transcription termination sequences. Therefore, all genes areequally integrated into the host cell genome, including the fusion geneof the therapeutic protein and either the HC or LC IgG gene.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1. A composition comprising a neurotherapeutic agent covalently linkedto a structure that is capable of crossing the blood brain barrier(BBB), wherein the composition is capable of producing an averageelevation of concentration in the brain of the neurotherapeutic agent ofat least about 5 ng/gram brain following peripheral administration. 2.The composition of claim 1 wherein the neurotherapeutic agent has amolecular weight greater than about 400 Daltons.
 3. The composition ofclaim 1 wherein the neurotherapeutic agent alone does not cross the BBBin a therapeutically effective amount following peripheraladministration.
 4. The composition of claim 1 wherein the structure thatis capable of crossing the BBB crosses the BBB on an endogenous BBBreceptor mediated transport system.
 5. The composition of claim 4wherein the endogenous BBB receptor mediated transport system isselected from the group consisting of the insulin receptor, transferrinreceptor, leptin receptor, lipoprotein receptor, and the IGF receptor.6. The composition of claim 5 wherein the endogenous BBB receptormediated transport system is the insulin BBB receptor mediated transportsystem.
 7. The composition of claim 1 wherein the structure that iscapable of crossing the BBB is an antibody.
 8. The composition of claim7 wherein the antibody is a monoclonal antibody (MAb).
 9. Thecomposition of claim 8 wherein the MAb is a chimeric MAb.
 10. Thecomposition of claim 9 wherein the chimeric antibody contains sufficienthuman sequences to avoid significant immunogenic reaction whenadministered to a human.
 11. The composition of claim 5 wherein thestructure that crosses the BBB on an endogenous BBB receptor mediatedtransport system is an antibody.
 12. The composition of claim 11 whereinthe antibody is a monoclonal antibody.
 13. The composition of claim 12wherein the MAb is a chimeric MAb.
 14. The composition of claim 13wherein the chimeric antibody contains sufficient human sequences toavoid significant immunogenic reaction when administered to a human. 15.The composition of claim 1 wherein the neurotherapeutic agent is aneurotrophin.
 16. The composition of claim 15 wherein the neurotrophinis selected from the group consisting of brain derived neurotrophicfactor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblastgrowth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-1 receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, and stem cell factor (SCF).
 17. The compositionof claim 15 wherein the neurotrophin comprises brain derivedneurotrophic factor (BDNF).
 18. The composition of claim 17 wherein theBDNF is a variant of native BDNF.
 19. The composition of claim 17wherein the BDNF is a two amino acid carboxyl-truncated variant.
 20. Thecomposition of claim 17 or 19 wherein the BDNF is a human BDNF.
 21. Thecomposition of claim 19 wherein the BDNF comprises a sequence that is atleast about 80% identical to the sequence of amino acids 466-582 of SEQID NO:
 24. 22. The composition of claim 1 wherein the neurotherapeuticagent is a neurotrophin and the structure that is capable of crossingthe blood brain barrier (BBB) is a MAb to an endogenous BBB receptormediated transport system.
 23. The composition of claim 22 wherein theneurotrophin is BDNF.
 24. The composition of claim 23 wherein the MAb isan antibody to the insulin BBB receptor mediated transport system. 25.The composition of claim 24 wherein the BDNF is a two amino acidcarboxy-truncated BDNF.
 26. The composition of claim 25 wherein the MAbis a chimeric MAb
 27. The composition of claim 26 wherein the chimericantibody contains sufficient human sequences to avoid significantimmunogenic reaction when administered to a human.
 28. The compositionof claim 26 wherein the insulin receptor is a human insulin receptor andwherein the BDNF is a human BDNF.
 29. The composition of claim 28wherein the BDNF comprises a sequence that is at least about 80%identical to the sequence of amino acids 466-582 of SEQ ID NO:
 24. 30.The composition of claim 29 wherein the BDNF is covalently linked at itsamino terminus to the carboxy terminus of the heavy chain of the MAb.31. The compostion of claim 29 wherein the BDNF is covalently linked atits amino terminus to the carboxy terminus of the light chain of theMAb.
 32. The composition of claim 30 wherein the heavy chain of the MAbcomprises a sequence that is at least about 80% identical to amino acids20-462 of SEQ ID NO:
 24. 33. The composition of claim 32 furthercomprising a linker between the heavy chain of the MAb and the BDNF. 34.The composition of claim 33 wherein the linker is S-S-M.
 35. Thecomposition of claim 34 further comprising the light chain of the MAb.36. The composition of claim 35 wherein the light chain comprises asequence that is at least about 80% identical to amino acids 21-234 ofSEQ ID NO:
 36. 37. The composition of claim 36 wherein the MAb isglycosylated.
 38. A pharmaceutical composition comprising thecomposition of claim 1, 18, 21, 30, or 31 and a pharmaceuticallyacceptable excipient.
 39. A composition comprising the composition ofclaim 1 and second composition comprising a second neurotherapeuticagent covalently linked to a second structure that is capable ofcrossing the blood brain barrier (BBB).
 40. The composition of claim 39wherein the first and second neurotherapeutic agents are different andthe first and second structures capable of crossing the BBB are the samestructure.
 41. The composition of claim 40 wherein the structure capableof crossing the BBB is an antibody comprising a first heavy chain and asecond heavy chain.
 42. The composition of claim 40 wherein the firstneurotherapeutic agent is covalently linked to the first heavy chain ofthe antibody and the second neurotherapeutic agent is covalently linkedto the second heavy chain of the antibody.
 43. A composition comprisingan agent covalently linked to a chimeric MAb to the human BBB insulinreceptor, wherein the MAb comprises a heavy chain and a light chain. 44.The composition of claim 43 wherein the agent is a therapeutic agent.45. The composition of claim 44 wherein the therapeutic agent is aneurotrophin.
 46. The composition of claim 45 wherein the neurotrophinis a BDNF.
 47. The composition of claim 46 wherein the agent is a twoamino acid carboxyl-terminal truncated BDNF.
 48. The composition ofclaim 46 wherein the heavy chain of the MAb is covalently linked to theBDNF to form a fusion protein, and wherein the sequence of said fusionprotein comprises a first sequence that is at least about 80% identicalto a sequence comprising amino acids 20-462 of SEQ ID NO: 24 and furthercomprises a second sequence that is at least about 80% identical to asequence comprising amino acids 466-582 of SEQ ID NO:
 24. 49. Thecomposition of claim 48 wherein the light chain of the MAb comprises asequence that is at least about 80% identical to a sequence comprisingamino acids 21-234 of SEQ ID NO:
 36. 50. The composition of claim 48wherein the MAb is glycosylated.
 51. The composition of claim 49 furthercomprising a peptide linker between the carboxyl terminus of the firstsequence and the amino terminus of the second sequence.
 52. Thecomposition of claim 51 wherein the linker comprises S-S-M.
 53. Thecomposition of claim 52 wherein the MAb is glycosylated.
 54. Acomposition for treating a neurological disorder comprising a BDNFcovalently linked to an immunoglobulin that is capable of crossing theblood brain barrier, wherein the composition is capable of crossing theBBB in an amount that is effective in treating the neurologicaldisorder.
 55. A fusion protein comprising: (i) a structure capable ofcrossing the BBB, covalently linked to (ii) a peptide that is active inthe central nervous system (CNS), wherein the structure capable ofcrossing the blood brain barrier and the peptide that is active in thecentral nervous system each retain an average of at least about 40% oftheir. activities, compared to their activities as separate entities.56. The fusion protein of claim 55 wherein the structure capable ofcrossing the blood brain barrier crosses the BBB on an endogenous BBBreceptor-mediated transporter.
 57. The fusion protein of claim 56wherein the endogenous BBB receptor mediated transport system isselected from the group consisting of the insulin receptor, transferrinreceptor, leptin receptor, lipoprotein receptor, and the IGF receptor.58. The fusion protein of claim 57 wherein the endogenous BBBreceptor-mediated transporter is selected from the group consisting ofthe insulin transporter and the transferrin transporter.
 59. The fusionprotein of claim 58 wherein the endogenous BBB receptor-mediatedtransporter is the insulin transporter.
 60. The fusion protein of claim59 wherein the insulin transporter is the human insulin transporter. 61.The fusion protein of claim 55 wherein the structure capable of crossingthe BBB is an antibody.
 62. The fusion protein of claim 61 wherein theantibody is a MAb.
 63. The fusion protein of claim 62 wherein the MAb isa chimeric MAb.
 64. The fusion protein of claim 61 wherein the antibodyis an antibody to an endogenous BBB receptor-mediated transporter 65.The fusion protein of claim 64 wherein the the endogenous BBB receptormediated transport system is selected from the group consisting of theinsulin receptor, transferrin receptor, leptin receptor, lipoproteinreceptor, and the IGF receptor.
 66. The fusion protein of claim 65wherein the endogenous BBB receptor-mediated transporter is selectedfrom the group consisting of the insulin transporter and the transferrintransporter.
 67. The fusion protein of claim 66 wherein the endogenousBBB receptor-mediated transporter is the insulin transporter.
 68. Thefusion protein of claim 67 wherein the insulin transporter is the humaninsulin transporter.
 69. The fusion protein of claim 55 wherein thepeptide that is active in the CNS is a neurotherapeutic agent.
 70. Thefusion protein of claim 69 wherein the neurotherapeutic agent is aneurotrophin.
 71. The fusion protein of claim 70 wherein theneurotrophin is selected from the group consisting of brain derivedneurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-4/5,fibroblast growth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erytropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-1 receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, and stem cell factor (SCF).
 72. The fusionprotein of claim 71 wherein the neurotrophin is BDNF.
 73. The fusionprotein of claim 72 wherein the BDNF is a truncated BDNF.
 74. The fusionprotein of claim 73 wherein the truncated BDNF is a carboxyl-truncatedBDNF.
 75. The fusion protein of claim 74 wherein the carboxyl-truncatedBDNF is lacking the two carboxyl terminal amino acids.
 76. The fusionprotein of claim 55 wherein the structure capable of crossing the BBBand the neurotherapeutic agent are covalently linked by a peptidelinker.
 77. A method of transport of an agent active in the CNS from theperipheral circulation across the BBB in an effective amount, comprisingperipherally administering to an individual the agent covalentlyattached to a structure that crosses the BBB, under conditions where theagent covalently attached to a structure that crosses the BBB istransported across the BBB in an effective amount.
 78. The method ofclaim 77 wherein the agent is a neurotherapeutic agent.
 79. A method fortreating a CNS disorder in an individual comprising peripherallyadministering to the individual an effective amount of a compositioncomprising a neurotherapeutic agent covalently attached to a structurecapable of crossing the BBB.
 80. The method of claim 79 wherein thestructure capable of crossing the BBB comprises an antibody to aninsulin receptor and the therapeutic agent comprises a BDNF.
 81. Themethod of claim 80 wherein the administering is selected from the groupconsisting of oral, intravenous, intramuscular, subcutaneous,intraperitoneal, rectal, transbuccal, intranasal, transdermal, andinhalation administering
 82. The method of claim 81 wherein theadministering is intravenous, intramuscular, or subcutaneous.
 83. Themethod of claim 79 wherein the CNS disorder is an acute CNS disorder.84. The method of claim 83 wherein the acute CNS disorder is selectedfrom the group consisting of spinal cord injury, brain injury focalbrain ischemia and global brain ischemia.
 85. The method of claim 83wherein the composition is administered only once.
 86. The method ofclaim 83 wherein the composition is administered at a frequency of nogreater than about once per week.
 87. The method of claim 79 wherein theCNS disorder is a chronic disorder.
 88. The method of claim 87 whereinthe chronic disorder is selected from the group consisting of chronicneurodegenerative disease, retinal ischemia, and depression.
 89. Themethod of claim 88 wherein the chronic neurodegenerative disease isselected from the group consisting of prion diseases, Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis,Huntington's disease, multiple sclerosis, transverse myelitis, motorneuron disease, Pick's disease, tuberous sclerosis, lysosomal storagedisorders, Canavan's disease, Rett's syndrome, spinocerebellar ataxias,Friedreich's ataxia, optic atrophy, and retinal degeneration.
 90. Themethod of claim 79 wherein the individual is a human.
 91. The method ofclaim 90 wherein the individual is administered a dose of thecomposition that is about 1 to about 100 mg.
 92. A compositioncomprising a cationic therapeutic peptide covalently linked to animmunoglobulin, wherein the cationic therapeutic peptide in thecomposition has a serum half-life that is an average of at least about5-fold greater than the serum half-life of the cationic therapeuticpeptide alone.
 93. The composition of claim 92 wherein the cationictherapeutic peptide comprises a neurotherapeutic agent.
 94. Thecomposition of claim 93 wherein the neurotherapeutic agent is aneurotrophin.
 95. The composition of claim 94 wherein the neurotrophinis selected from the group consisting of brain derived neurotrophicfactor (BDNF), nerve growth factor (NGF), neurotrophin-4/5, fibroblastgrowth factor (FGF)-2 and other FGFs, neurotrophin (NT)-3,erythropoietin (EPO), hepatocyte growth factor (HGF), epidermal growthfactor (EGF), transforming growth factor (TGF)-α, TGF-β, vascularendothelial growth factor (VEGF), interleukin-1 receptor antagonist(IL-1ra), ciliary neurotrophic factor (CNTF), glial-derived neurotrophicfactor (GDNF), neurturin, platelet-derived growth factor (PDGF),heregulin, neuregulin, artemin, persephin, interleukins,granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF,netrins, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF),midkine, pleiotrophin, bone morphogenetic proteins (BMPs), netrins,saposins, semaphorins, and stem cell factor (SCF).
 96. The compositionof claim 95 wherein the neurotrophin is BDNF.
 97. The composition ofclaim 92 wherein the immunoglobulin is an antibody to an endogenous BBBreceptor-mediated transport system.